U.S. patent application number 12/132320 was filed with the patent office on 2009-05-07 for phase-sensitively detected reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins.
This patent application is currently assigned to The Research Foundation of State University of New York. Invention is credited to Seho Kim, Thomas A. Szyperski.
Application Number | 20090115414 12/132320 |
Document ID | / |
Family ID | 31188570 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090115414 |
Kind Code |
A1 |
Szyperski; Thomas A. ; et
al. |
May 7, 2009 |
PHASE-SENSITIVELY DETECTED REDUCED DIMENSIONALITY NUCLEAR MAGNETIC
RESONANCE SPECTROSCOPY FOR RAPID CHEMICAL SHIFT ASSIGNMENT AND
SECONDARY STRUCTURE DETERMINATION OF PROTEINS
Abstract
The present invention discloses eleven reduced dimensionality
(RD) triple resonance nuclear magnetic resonance (NMR) experiments
for measuring chemical shift values of certain nuclei in a protein
molecule, where the chemical shift values encoded in a peak pair of
an NMR spectrum are detected in a phase sensitive manner. The RD 3D
HA,CA,(CO),N,HN NMR and RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiments
are designed to yield "sequential" connectivities, while the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta., CO,HA NMR and RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiments
provide "intraresidue" connectivities. The RD 3D H,C,C,H-COSY NMR,
RD 3D H,C,C,H-TOCSY NMR, and RD 2D H,C,H-COSY NMR experiments allow
one to obtain assignments for aliphatic and aromatic side chain
chemical shifts, while the RD 2D HB,CB,(CG,CD),HD NMR experiment
provide information for the aromatic side chain chemical shifts. In
addition, methods of conducting suites of RD triple resonance NMR
experiments for high-throughput resonance assignment of proteins
and determination of secondary structure elements are
disclosed.
Inventors: |
Szyperski; Thomas A.;
(Amherst, NY) ; Kim; Seho; (Highland Park,
NJ) |
Correspondence
Address: |
NIXON PEABODY LLP - PATENT GROUP
1100 CLINTON SQUARE
ROCHESTER
NY
14604
US
|
Assignee: |
The Research Foundation of State
University of New York
|
Family ID: |
31188570 |
Appl. No.: |
12/132320 |
Filed: |
June 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10628818 |
Jul 28, 2003 |
7396685 |
|
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12132320 |
|
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60399332 |
Jul 26, 2002 |
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Current U.S.
Class: |
324/309 |
Current CPC
Class: |
Y10T 436/24 20150115;
G01R 33/4633 20130101; G01R 33/465 20130101 |
Class at
Publication: |
324/309 |
International
Class: |
G01R 33/44 20060101
G01R033/44 |
Goverment Interests
[0002] This invention arose out of research sponsored by the
National Science Foundation (Grant No. MCB 0075773) and National
Institutes of Health (Grant No. P50 GM62413-01). The U.S.
Government may have certain rights in this invention.
Claims
1. A method of conducting a reduced dimensionality
three-dimensional (3D) HA,CA,(CO),N,HN nuclear magnetic resonance
(NMR) experiment by measuring the chemical shift values for the
following nuclei of a protein molecule having two consecutive amino
acid residues, i-1 and i: (1) an .alpha.-proton of amino acid
residue i-1, .sup.1H.sup..alpha..sub.i-1; (2) an .alpha.-carbon of
amino acid residue i-1, .sup.13C.sup..alpha..sub.i-1; (3) a
polypeptide backbone amide nitrogen of amino acid residue i,
.sup.15N.sub.i; and (4) a polypeptide backbone amide proton of
amino acid residue i, .sup.1H.sup.N.sub.i, wherein the chemical
shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 which are encoded in a peak pair of a
3D NMR spectrum are detected in a phase sensitive manner, said
method comprising: providing a protein sample; applying
radiofrequency pulses to the protein sample which effect a nuclear
spin polarization transfer wherein the chemical shift evolutions of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 of
amino acid residue i-1 are connected to the chemical shift
evolutions of .sup.15N.sub.i and .sup.1H.sup.N.sub.i of amino acid
residue i, under conditions effective (1) to generate NMR signals
encoding the chemical shift values of .sup.13C.sup..alpha..sub.i-1
and .sup.15N.sub.i in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup..alpha.) and
t.sub.2(.sup.15N), respectively, and the chemical shift value of
.sup.1H.sup.N.sub.i in a direct time domain dimension,
t.sub.3(.sup.1H.sup.N), and (2) to sine modulate the
.sup.13C.sup..alpha..sub.i-1 chemical shift evolution in
t.sub.1(.sup.13C.sup..alpha.) with the chemical shift evolution of
.sup.1H.sup..alpha..sub.i-1; and processing the NMR signals to
generate a sine-modulated 3D NMR spectrum with an anti-phase peak
pair derived from said sine modulating, wherein (1) the chemical
shift values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i are measured
in two frequency domain dimensions, .omega..sub.2(.sup.15N) and
.omega..sub.3(.sup.1H.sup.N), respectively, and (2) the chemical
shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha.), by the frequency
difference between the two peaks forming said anti-phase peak pair
and the frequency at the center of the two peaks, respectively,
wherein said sine-modulated 3D NMR spectrum enables detection of
the chemical shift value of .sup.1H.sup..alpha..sub.i-1 in a phase
sensitive manner.
2. The method according to claim 1, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i-1 and .sup.15N.sub.i in a
phase sensitive manner in t.sub.1(.sup.13C.sup..alpha.) and
t.sub.2(.sup.15N), respectively, and the chemical shift value of
.sup.1H.sup.N.sub.i in t.sub.3(.sup.1H.sup.N) and (2) to cosine
modulate the .sup.13C.sup..alpha..sub.i-1 chemical shift evolution
in t.sub.1(.sup.13C.sup..alpha.) with the chemical shift evolution
of .sup.1H.sup..alpha..sub.i-1 for the additional NMR signals, and
said processing the NMR signals and the additional NMR signals
further comprises generating a cosine-modulated 3D NMR spectrum
with an in-phase peak pair derived from said cosine modulating, a
sum 3D NMR spectrum generated by adding said sine-modulated 3D NMR
spectrum and said cosine-modulated 3D NMR spectrum, and a
difference 3D NMR spectrum generated by subtracting said
cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR
spectrum, wherein combined use of said sum 3D NMR spectrum and said
difference 3D NMR spectrum enables placement of the two peaks
forming said peak pairs into separate spectra, thereby allowing
phase-sensitive editing of the two peaks forming said peak
pairs.
3. The method according to claim 1, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of .sup.15N.sub.i does not occur and said processing the
NMR signals generates a two dimensional (2D) NMR spectrum with a
peak pair wherein (1) the chemical shift value of
.sup.1H.sup.N.sub.i is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.N), and (2) the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha.), by the frequency difference
between the two peaks forming said peak pair and the frequency at
the center of the two peaks, respectively.
4. The method according to claim 1, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of a polypeptide backbone carbonyl carbon of amino acid
residue i-1, .sup.13C'.sub.i-1, occurs under conditions effective
to generate NMR signals encoding the chemical shift value of
.sup.13C'.sub.i-1 in a phase sensitive manner in an indirect time
domain dimension, t.sub.4(.sup.13C'), and said processing the NMR
signals generates a four dimensional (4D) NMR spectrum with a peak
pair wherein (1) the chemical shift values of .sup.15N.sub.i,
.sup.1H.sup.N.sub.i and .sup.13C'.sub.i-1 are measured in three
frequency domain dimensions, .omega..sub.2(.sup.15N),
.omega..sub.3(.sup.1H.sup.N), and .omega..sub.4(.sup.13C'),
respectively, and (2) the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha.), by the frequency difference
between the two peaks forming said peak pair and the frequency at
the center of the two peaks, respectively.
5. The method according to claim 1, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i-1 and .sup.15N.sub.i in a
phase sensitive manner in t.sub.1(.sup.13C.sup..alpha.) and
t.sub.2(.sup.15N), respectively, and the chemical shift value of
.sup.1H.sup.N.sub.i in t.sub.3(.sup.1H.sup.N), and (2) to avoid
sine modulating the .sup.13C.sup..alpha..sub.i-1 chemical shift
evolution in t.sub.1(.sup.13C.sup..alpha.) with the chemical shift
evolution of .sup.1H.sup..alpha..sub.i-1 for the additional NMR
signals, and said processing the NMR signals and the additional NMR
signals generates a 3D NMR spectrum with an additional peak located
centrally between two peaks forming said peak pair which measures
the chemical shift value of .sup.13C.sup..alpha..sub.i-1 along
.omega..sub.1(.sup.13C.sup..alpha.).
6. The method according to claim 5, wherein said additional peak is
derived from .sup.13C.sup..alpha. nuclear spin polarization.
7. The method according to claim 6, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2B, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup..alpha..sub.i-1 magnetization, which
is transferred to .sup.13C.sup..alpha..sub.i-1, to .sup.15N.sub.i,
and to .sup.1H.sup.N.sub.i, to generate the NMR signal.
8. The method according to claim 7, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3B to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3B,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pair and a second NMR
subspectrum derived from said adding which contains said additional
peak located centrally between the two peaks forming said peak
pair.
9. A method of conducting a reduced dimensionality
three-dimensional (3D) H,C,-(C-TOCSY-CO),N,HN nuclear magnetic
resonance (NMR) experiment by measuring the chemical shift values
for the following nuclei of a protein molecule having two
consecutive amino acid residues, i-1 and i: (1) aliphatic protons
of amino acid residue i-1, .sup.1H.sup.ali.sub.i-1; (2) aliphatic
carbons of amino acid residue i-1, .sup.13C.sup.ali.sub.i-1; (3) a
polypeptide backbone amide nitrogen of amino acid residue i,
.sup.15N.sub.i; and (4) a polypeptide backbone amide proton of
amino acid residue i, .sup.1H.sup.N.sub.i, wherein the chemical
shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 which are encoded in peak pairs of a 3D
NMR spectrum are detected in a phase sensitive manner, said method
comprising: providing a protein sample; applying radiofrequency
pulses to the protein sample which effect a nuclear spin
polarization transfer wherein the chemical shift evolutions of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 of amino acid
residue i-1 are connected to the chemical shift evolutions of
.sup.15N.sub.i and .sup.1H.sup.N.sub.i of amino acid residue i,
under conditions effective (1) to generate a NMR signal encoding
the chemical shift values of .sup.13C.sup.ali.sub.i-1 and
.sup.15N.sub.i in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup.ali) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i
in a direct time domain dimension, t.sub.3(.sup.1H.sup.N), and (2)
to sine modulate the chemical shift evolutions of
.sup.13C.sup.ali.sub.i-1 in t.sub.1(.sup.13C.sup.ali) with the
chemical shift evolutions of .sup.1H.sup.ali.sub.i-1; and
processing the NMR signals to generate a sine-modulated 3D NMR
spectrum with anti-phase peak pairs derived from said sine
modulating wherein (1) the chemical shift values of .sup.15N.sub.i
and .sup.1H.sup.N.sub.i are measured in two frequency domain
dimensions, .omega..sub.2(.sup.15N) and
.omega..sub.3(.sup.1H.sup.N), respectively, and (2) the chemical
shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.ali), by the frequency
differences between each of the two peaks forming each of said
anti-phase peak pairs and the frequencies at the center of the two
peaks, respectively, wherein said sine-modulated 3D NMR spectrum
enables detection of the chemical shift value of
.sup.1H.sup.ali.sub.i-1 in a phase sensitive manner.
10. The method according to claim 9, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup.ali.sub.i-1 and .sup.15N.sub.i in a phase
sensitive manner in t.sub.1(.sup.13C.sup.ali) and
t.sub.2(.sup.15N), respectively, and the chemical shift value of
.sup.1H.sup.N.sub.i in t.sub.3(.sup.1H.sup.N) and (2) to cosine
modulate the chemical shift evolutions of .sup.13C.sup.ali.sub.i-1
in t.sub.1(.sup.13C.sup.ali) with the chemical shift evolutions of
.sup.1H.sup.ali.sub.i-1 for the additional NMR signals, and said
processing the NMR signals and the additional NMR signals further
comprises generating a cosine-modulated 3D NMR spectrum with
in-phase peak pairs derived from said cosine modulating, a sum 3D
NMR spectrum generated by adding said sine-modulated 3D NMR
spectrum and said cosine-modulated 3D NMR spectrum, and a
difference 3D NMR spectrum generated by subtracting said
cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR
spectrum, wherein combined use of said sum 3D NMR spectrum and said
difference 3D NMR spectrum enables placement of the two peaks
forming said peak pairs into separate spectra, thereby allowing
phase-sensitive editing of the two peaks forming said peak
pairs.
11. The method according to claim 9, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of .sup.15N.sub.i does not occur and said processing the
NMR signals generates a two dimensional (2D) NMR spectrum with peak
pairs wherein (1) the chemical shift value of .sup.1H.sup.N.sub.i
is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.N), and (2) the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 are measured
in a frequency domain dimension, .omega..sub.1(.sup.13C.sup.ali),
by the frequency differences between the two peaks forming said
peak pairs and the frequencies at the center of the two peaks,
respectively.
12. The method according to claim 9, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of a polypeptide backbone carbonyl carbon of amino acid
residue i-1, .sup.13C'.sub.i-1, occurs under conditions effective
to generate NMR signals encoding the chemical shift value of
.sup.13C'.sub.i-1 in a phase sensitive manner in an indirect time
domain dimension, t.sub.4(.sup.13C'), and said processing the NMR
signals generates a four dimensional (4D) NMR spectrum with variant
peak pairs wherein (1) the chemical shift values of .sup.15N.sub.i,
.sup.1H.sup.N.sub.i and .sup.13C'.sub.i-1 are measured in three
frequency domain dimensions, .omega..sub.2(.sup.15N),
.omega..sub.3(.sup.1H.sup.N), and .omega..sub.4(.sup.13C'),
respectively, and (2) the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 are measured
in a frequency domain dimension, .omega..sub.1(.sup.13C.sup.ali),
by the frequency differences between the two peaks forming said
variant peak pairs and the frequencies at the center of the two
peaks, respectively.
13. The method according to claim 9, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup.ali.sub.i-1 and .sup.15N.sub.i in a phase
sensitive manner in t.sub.1(.sup.13C.sup.ali) and t.sub.2(.sup.15N)
and the chemical shift value of .sup.1H.sup.N.sub.i in
t.sub.3(.sup.1H.sup.N), and (2) to avoid sine modulating the
chemical shift evolutions of .sup.13C.sup.ali.sub.i-1 in
t.sub.1(.sup.13C.sup.ali) with the chemical shift evolution of
.sup.1H.sup..alpha..sub.i-1 for the additional NMR signals, and
said processing the NMR signals and the additional NMR signals
generates a 3D NMR spectrum with additional peaks located centrally
between said peak pairs which measure the chemical shift values of
.sup.13C.sup.ali.sub.i-1 along .omega..sub.1(.sup.13C.sup.ali).
14. The method according to claim 13, wherein said additional peaks
are derived from .sup.13C.sup.ali nuclear spin polarization.
15. The method according to claim 14, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2C, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup.ali.sub.i-1 magnetization, and
.sup.1H.sup.ali.sub.i-1 magnetization is transferred to
.sup.13C.sup.ali.sub.i-1, to .sup.13C.sup..alpha..sub.i-1, to
.sup.13C'.sub.i-1, to .sup.15N.sub.i, and to .sup.1H.sup.N.sub.i,
where the NMR signal is detected.
16. The method according to claim 15, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3C to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3C,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pairs, and a second NMR
subspectrum derived from said adding which contains said additional
peaks located centrally between said peak pairs.
17. A method of conducting a reduced dimensionality
three-dimensional (3D)
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA nuclear magnetic
resonance (NMR) experiment by measuring the chemical shift values
for the following nuclei of a protein molecule having an amino acid
residue, i: (1) a .beta.-proton of amino acid residue i,
.sup.1H.sup..beta..sub.i; (2) a .beta.-carbon of amino acid residue
i, .sup.13C.sup..beta..sub.i; (3) an .alpha.-proton of amino acid
residue i, .sup.1H.sup..alpha..sub.i; (4) an .alpha.-carbon of
amino acid residue i, .sup.13C.sup..alpha..sub.i; and (5) a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i, wherein the chemical shift values of
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i/.sup.13C.sup..beta..sub.i which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner, said method comprising: providing a protein
sample; applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer wherein the chemical
shift evolutions of .sup.1H.sup..alpha..sub.i,
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are connected to the chemical shift
evolution of .sup.13C'.sub.i, under conditions effective (1) to
generate NMR signals encoding the chemical shift values of
.sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.13C'.sub.i in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup..alpha./.beta.) and
t.sub.2(.sup.13C'), respectively, and the chemical shift value of
.sup.1H.sup..alpha..sub.i in a direct time domain dimension,
t.sub.3(.sup.1H.sup..alpha.) and (2) to sine modulate the chemical
shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i, respectively; and processing the NMR
signals to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from said sine modulating wherein (1)
the chemical shift values of .sup.13C'.sub.i and
.sup.1H.sup..alpha..sub.i are measured in two frequency domain
dimensions, .omega..sub.2(.sup.13C') and
.omega..sub.3(.sup.1H.sup..alpha.), respectively, and (2) (i) the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequency differences between each of the two peaks forming each of
said anti-phase peak pairs, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming said anti-phase peak pairs,
wherein said sine-modulated 3D NMR spectrum enables detection of
the chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i in a phase sensitive manner.
18. The method according to claim 17, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.13C'.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.13C'),
respectively, and the chemical shift value of
.sup.1H.sup..alpha..sub.i in t.sub.3(.sup.1H.sup..alpha.) and (2)
to cosine modulate the .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i chemical shift evolutions in
t.sub.1(.sup.13C.sup..alpha./.beta.) with the chemical shift
evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals and said
processing the NMR signals and the additional NMR signals further
comprises generating a cosine-modulated 3D NMR spectrum with
in-phase peak pairs derived from said cosine modulating, a sum 3D
NMR spectrum generated by adding said sine-modulated 3D NMR
spectrum and said cosine-modulated 3D NMR spectrum, and a
difference 3D NMR spectrum generated by subtracting said
cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR
spectrum, wherein combined use of said sum 3D NMR spectrum and said
difference 3D NMR spectrum enables placement of the two peaks
forming said peak pairs into separate spectra, thereby allowing
phase-sensitive editing of the two peaks forming said peak
pairs.
19. The method according to claim 17, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of .sup.13C'.sub.i does not occur and said processing the
NMR signals generates a two dimensional (2D) NMR spectrum with peak
pairs wherein (1) the chemical shift value of
.sup.1H.sup..alpha..sub.i is measured in a frequency domain
dimension, .omega..sub.2(.sup.1H.sup..alpha.), and (2) (i) the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequency differences between two peaks forming said peak pairs,
respectively, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i, and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming said peak pairs.
20. The method according to claim 17 wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.15N.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N) and the
chemical shift value of .sup.1H.sup..alpha..sub.i in
t.sub.3(.sup.1H.sup..alpha.), and (2) to avoid sine modulating the
chemical shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signal, and said
processing the NMR signals and the additional NMR signals generates
a 3D NMR spectrum with additional peaks located centrally between
the two peaks forming said peak pairs which measure the chemical
shift values of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i along
.omega..sub.1(.sup.13C.sup..alpha./.beta.).
21. The method according to claim 20, wherein said additional peaks
are derived from .sup.13C.sup..alpha. and .sup.13C.sup..beta.
nuclear spin polarization.
22. The method according to claim 21, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2E, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i magnetization, and
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i polarization
is transferred to .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i, to .sup.13C'.sub.i, and back to
.sup.1H.sup..alpha..sub.i, where the NMR signal is detected.
23. The method according to claim 22, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3E to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3E,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pairs, and a second NMR
subspectrum derived from said adding which contains said additional
peaks located centrally between the two peaks forming said peak
pairs.
24. A method of conducting a reduced dimensionality
three-dimensional (3D)
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN nuclear magnetic
resonance (NMR) experiment by measuring the chemical shift values
for the following nuclei of a protein molecule having an amino acid
residue, i: (1) a .beta.-proton of amino acid residue i,
.sup.1H.sup..beta..sub.i; (2) a .beta.-carbon of amino acid residue
i, .sup.13C.sup..beta..sub.i; (3) an .alpha.-proton of amino acid
residue i, .sup.1H.sup..alpha..sub.i; (4) an .alpha.-carbon of
amino acid residue i, .sup.13C.sup..alpha..sub.i; (5) a polypeptide
backbone amide nitrogen of amino acid residue i, .sup.15N.sub.i;
and (6) a polypeptide backbone amide proton of amino acid residue
i, .sup.1H.sup.N.sub.i, wherein the chemical shift values of
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i/.sup.13C.sup..beta..sub.i which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner, said method comprising: providing a protein
sample; applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer wherein the chemical
shift evolutions of .sup.1H.sup..alpha..sub.i,
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are connected to the chemical shift
evolutions of .sup.15N.sub.i and .sup.1H.sup.N.sub.i, under
conditions effective (1) to generate NMR signals encoding the
chemical shift values of .sup.13C.sup..alpha..sub.i,
.sup.13C.sup..beta..sub.i and .sup.15N.sub.i in a phase sensitive
manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i
in a direct time domain dimension, t.sub.3(.sup.1H.sup.N) and (2)
to sine modulate the chemical shift evolutions of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i in
t.sub.1(.sup.13C.sup..alpha./.beta.) with the chemical shift
evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i, respectively; and processing the NMR
signals to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from said sine modulating wherein (1)
the chemical shift values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i
are measured in two frequency domain dimensions,
.omega..sub.2(.sup.15N) and .omega..sub.3(.sup.1H.sup.N),
respectively, and (2) (i) the chemical shift values of
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i are measured
in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequency
differences between each of the two peaks forming each of said
anti-phase peak pairs, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of said two peaks forming said anti-phase peak pairs,
wherein said sine-modulated 3D NMR spectrum enables detection of
the chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i in a phase sensitive manner.
25. The method according to claim 24, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.15N.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i
in t.sub.3(.sup.1H.sup.N) and (2) to cosine modulate the
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i chemical
shift evolutions in t.sub.1(.sup.13C.sup..alpha./.beta.) with the
chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals, and said
processing the NMR signals and the additional NMR signals further
comprises generating a cosine-modulated 3D NMR spectrum with
in-phase peak pairs derived from said cosine modulating, a sum 3D
NMR spectrum generated by adding said sine-modulated 3D NMR
spectrum and said cosine-modulated 3D NMR spectrum, and a
difference 3D NMR spectrum generated by subtracting said
cosine-modulated 3D NMR spectrum from said sine-modulated 3D NMR
spectrum, wherein combined use of said sum 3D NMR spectrum and said
difference 3D NMR spectrum enables placement of the two peaks
forming said peak pairs into separate spectra, thereby allowing
phase-sensitive editing of the two peaks forming said peak
pairs.
26. The method according to claim 24, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of .sup.15N.sub.i does not occur and said processing the
NMR signals generates a two dimensional (2D) NMR spectrum with peak
pairs wherein (1) the chemical shift value of .sup.1H.sup.N.sub.i
is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.N), and (2) (i) the chemical shift values
of .sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequency
differences between the two peaks forming said peak pairs, and (ii)
the chemical shift values of .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequencies at the center of the two peaks forming said peak
pairs.
27. The method according to claim 24, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.15N.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N) and the
chemical shift value of .sup.1H.sup.N.sub.i in
t.sub.3(.sup.1H.sup.N), and (2) to avoid sine modulating the
chemical shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals, and said
processing the NMR signals and the additional NMR signals generates
a 3D NMR spectrum with additional peaks located centrally between
the two peaks forming said peak pairs which measure the chemical
shift values of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i along
.omega..sub.1(.sup.13C.sup..alpha./.beta.).
28. The method according to claim 27, wherein said additional peaks
are derived from .sup.13C.sup..alpha. and .sup.13C.sup..beta.
nuclear spin polarization.
29. The method according to claim 28, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2F, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i magnetization, and
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i
magnetization is transferred to .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i, to .sup.15N.sub.i, and to
.sup.1H.sup.N.sub.i, where the NMR signal is detected.
30. The method according to claim 29, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3F to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3F,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pairs, and a second NMR
subspectrum derived from said adding which contains said additional
peaks located centrally between the two peaks forming said peak
pairs.
31. A method of conducting a reduced dimensionality
three-dimensional (3D) H,C,C,H-COSY nuclear magnetic resonance
(NMR) experiment by measuring the chemical shift values for
.sup.1H.sup.m, .sup.13C.sup.m, .sup.1H.sup.n, and .sup.13C.sup.n of
a protein molecule wherein m and n indicate atom numbers of two CH,
CH.sub.2 or CH.sub.3 groups that are linked by a single covalent
carbon-carbon bond in an amino acid residue, wherein the chemical
shift values of .sup.1H.sup.m and .sup.13C.sup.m which are encoded
in a peak pair of a 3D NMR spectrum are detected in a phase
sensitive manner, said method comprising: providing a protein
sample; applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer wherein the chemical
shift evolutions of .sup.1H.sup.m and .sup.13C.sup.m are connected
to the chemical shift evolutions of .sup.1H.sup.n and
.sup.13C.sup.m, under conditions effective (1) to generate NMR
signals encoding the chemical shift values of .sup.13C.sup.m and
.sup.13C.sup.n in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup.m) and
t.sub.2(.sup.13C.sup.n), respectively, and the chemical shift value
of .sup.1H.sup.n in a direct time domain dimension,
t.sub.3(.sup.1H.sup.n), and (2) to sine modulate the chemical shift
evolution of .sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the
chemical shift evolution of .sup.1H.sub.m; and processing the NMR
signals to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from said sine modulating wherein (1)
the chemical shift values of .sup.13C.sup.n and .sup.1H.sup.n are
measured in two frequency domain dimensions,
.omega..sub.2(.sup.13C.sup.n) and .omega..sub.3(.sup.1H.sup.n),
respectively, and (2) the chemical shift values of .sup.1H.sup.m
and .sup.13C.sup.m are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.m), by the frequency differences between
each of the two peaks forming each of said anti-phase peak pairs
and the frequencies at the center of the two peaks, respectively,
wherein said sine-modulated 3D NMR spectrum enables detection of
the chemical shift value of .sup.1H.sub.m in a phase sensitive
manner.
32. The method according to claim 31, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup.m and .sup.13C.sup.n in a phase sensitive
manner in t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n),
respectively, and the chemical shift value of .sup.1H.sup.n in
t.sub.3(.sup.1H.sup.n), and (2) to cosine modulate the
.sup.13C.sup.m chemical shift evolution in t.sub.1(.sup.13C.sup.m)
with the chemical shift evolution of .sup.1H.sub.m for the
additional NMR signals, and said processing the NMR signals and the
additional NMR signals further comprises generating a
cosine-modulated 3D NMR spectrum with in-phase peak pairs derived
from said cosine modulating, a sum 3D NMR spectrum generated by
adding said sine-modulated 3D NMR spectrum and said
cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum
generated by subtracting said cosine-modulated 3D NMR spectrum from
said sine-modulated 3D NMR spectrum, wherein combined use of said
sum 3D NMR spectrum and said difference 3D NMR spectrum enables
placement of the two peaks forming said peak pairs into separate
spectra, thereby allowing phase-sensitive editing of the two peaks
forming said peak pairs.
33. The method according to claim 31, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of .sup.13C.sup.n does not occur and said processing the
NMR signals generates a two dimensional (2D) NMR spectrum with peak
pairs wherein (1) the chemical shift value of .sup.1H.sup.n is
measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.n), and (2) the chemical shift values of
.sup.1H.sup.m and .sup.13C.sup.m are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.m), by the frequency
differences between the two peaks forming said peak pairs and the
frequencies at the center of the two peaks, respectively.
34. The method according to claim 31, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate an additional NMR signal encoding the chemical shift
values of .sup.13C.sup.m and .sup.13C.sup.n in a phase sensitive
manner in t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n) and
the chemical shift value of .sup.1H.sup.n in t.sub.3(.sup.1H), and
(2) to avoid sine modulating the chemical shift evolution of
.sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the chemical shift
evolution of .sup.1H.sup.m for the additional NMR signal, and said
processing the NMR signals and the additional NMR signal generates
a 3D NMR spectrum with additional peaks located centrally between
the two peaks forming said peak pairs which measure the chemical
shift value of .sup.13C.sup.m along
.omega..sub.1(.sup.13C.sup.m).
35. The method according to claim 34, wherein said additional peaks
are derived from .sup.13C.sup.m nuclear spin polarization.
36. The method according to claim 35, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2H, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup.m magnetization, and .sup.1H.sup.m
magnetization is transferred to .sup.13C.sup.m, to .sup.13C.sup.n,
and to .sup.1H.sup.n, where the NMR signal is detected.
37. The method according to claim 36, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3H to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3H,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pairs, and a second NMR
subspectrum derived from said adding which contains said additional
peaks located centrally between the two peaks forming said peak
pairs.
38. A method of conducting a reduced dimensionality
three-dimensional (3D) H,C,C,H-TOCSY nuclear magnetic resonance
(NMR) experiment by measuring the chemical shift values for
.sup.1H.sup.m, .sup.13C.sup.m, .sup.1H.sup.n, and .sup.13C.sup.n of
a protein molecule wherein m and n indicate atom numbers of two CH,
CH.sub.2 or CH.sub.3 groups that may or may not be directly linked
by a single covalent carbon-carbon bond in an amino acid residue,
wherein the chemical shift values of .sup.1H.sup.m and
.sup.13C.sup.m which are encoded in a peak pair of a 3D NMR
spectrum are detected in a phase sensitive manner, said method
comprising: providing a protein sample; applying radiofrequency
pulses to the protein sample which effect a nuclear spin
polarization transfer wherein the chemical shift evolutions of
.sup.1H.sup.m and .sup.13C.sup.m are connected to the chemical
shift evolutions of .sup.1H.sup.n and .sup.13C.sup.n, under
conditions effective (1) to generate NMR signals encoding the
chemical shift values of .sup.13C.sup.m and .sup.13C.sup.n in a
phase sensitive manner in two indirect time domain dimensions,
t.sub.1(.sup.13 C.sup.m) and t.sub.2(.sup.13C.sup.n), respectively,
and the chemical shift value of .sup.1H.sup.n in a direct time
domain dimension, t.sub.3(.sup.1H.sup.n), and (2) to sine modulate
the chemical shift evolution of .sup.13C.sup.m in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sup.m; and processing the NMR signals to generate a
sine-modulated 3D NMR spectrum with anti-phase peak pairs derived
from said sine modulating wherein (1) the chemical shift values of
.sup.13C.sup.n and .sup.1H.sup.n are measured in two frequency
domain dimensions, .omega..sub.2(.sup.13C.sup.n) and
.omega..sub.3(.sup.1H.sup.n), respectively, and (2) the chemical
shift values of .sup.1H.sup.m and .sup.13C.sup.m are measured in a
frequency domain dimension, .omega..sub.1(.sup.13C.sup.m), by the
frequency differences between each of the two peaks forming each of
said anti-phase peak pairs and the frequencies at the center of the
two peaks, respectively, wherein said sine-modulated 3D NMR
spectrum enables detection of the chemical shift value of
.sup.1H.sup.m and in a phase sensitive manner.
39. The method according to claim 38, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup.m and .sup.13C.sup.n in a phase sensitive
manner in t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n),
respectively, and the chemical shift value of .sup.1H.sup.n in
t.sub.3(.sup.1H.sup.n) and (2) to cosine modulate the
.sup.13C.sup.m chemical shift evolution in t.sub.1(.sup.13C.sup.m)
with the chemical shift evolution of .sup.1H.sub.m for the
additional NMR signals, and said processing the NMR signals and the
additional NMR signals further comprises generating a
cosine-modulated 3D NMR spectrum with in-phase peak pairs derived
from said cosine modulating, a sum 3D NMR spectrum generated by
adding said sine-modulated 3D NMR spectrum and said
cosine-modulated 3D NMR spectrum, and a difference 3D NMR spectrum
generated by subtracting said cosine-modulated 3D NMR spectrum from
said sine-modulated 3D NMR spectrum, wherein combined use of said
sum 3D NMR spectrum and said difference 3D NMR spectrum enables
placement of the two peaks forming said peak pairs into separate
spectra, thereby allowing phase-sensitive editing of the two peaks
forming said peak pairs.
40. The method according to claim 38, wherein said applying
radiofrequency pulses is carried out so that the chemical shift
evolution of .sup.13C.sup.n does not occur and said processing the
NMR signals generates a two dimensional (2D) NMR spectrum with peak
pairs wherein (1) the chemical shift value of .sup.1H.sup.n is
measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.n), and (2) the chemical shift values of
.sup.1H.sup.m and .sup.13C.sup.m are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.m), by the frequency
differences between the two peaks forming said peak pairs and the
frequencies at the center of the two peaks, respectively.
41. The method according to claim 38, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup.m and .sup.13C.sup.n in a phase sensitive
manner in t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n) and
the chemical shift value of .sup.1H.sup.n in
t.sub.3(.sup.1H.sup.n), and (2) to avoid sine modulating the
chemical shift evolution of .sup.13C.sup.m in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sup.m for the additional NMR signals, and said processing
the NMR signals and the additional NMR signals generates a 3D NMR
spectrum with additional peaks located centrally between the two
peaks forming said peak pairs which measure the chemical shift
value of .sup.13C.sup.m along .omega..sub.1(.sup.13C.sup.m).
42. The method according to claim 41, wherein said additional peaks
are derived from .sup.13C.sup.m nuclear spin polarization.
43. The method according to claim 42, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2I, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup.m magnetization, and .sup.1H.sup.m
magnetization is transferred to .sup.13C.sup.m, to .sup.13C.sup.n,
and to .sup.1H.sup.n, where the NMR signal is detected.
44. The method according to claim 43, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3I to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3I,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pairs, and a second NMR
subspectrum derived from said adding which contains said additional
peaks located centrally between the two peaks forming said peak
pairs.
45. A method of conducting a reduced dimensionality two-dimensional
(2D) HB,CB,(CG,CD),HD nuclear magnetic resonance NMR) experiment by
measuring the chemical shift values for the following nuclei of a
protein molecule: (1) a .beta.-proton of an amino acid residue with
an aromatic side chain, .sup.1H.sup..beta.; (2) a .beta.-carbon of
an amino acid residue with an aromatic side chain,
.sup.13C.sup..beta.; and (3) a .delta.-proton of an amino acid
residue with an aromatic side chain, .sup.1H.sup..delta., wherein
the chemical shift values of .sup.1H.sup..beta. and
.sup.13C.sup..beta. which are encoded in a peak pair of a 2D NMR
spectrum are detected in a phase sensitive manner, said method
comprising: providing a protein sample; applying radiofrequency
pulses to the protein sample which effect a nuclear spin
polarization transfer wherein the chemical shift evolutions of
.sup.1H.sup..beta. and .sup.13C.sup..beta. are connected to the
chemical shift evolution of .sup.1H.sup..delta., under conditions
effective (1) to generate NMR signals encoding the chemical shift
value of .sup.13C.sup..beta. in a phase sensitive manner in an
indirect time domain dimension, t.sub.1(.sup.13C.sup..beta.), and
the chemical shift value of .sup.1H.sup..delta. in a direct time
domain dimension, t.sub.2(.sup.1H.sup..delta.), and (2) to sine
modulate the chemical shift evolution of .sup.13C.sup..beta. in
t.sub.1(.sup.13C.sup..beta.) with the chemical shift evolution of
.sup.1H.sup..beta.; and processing the NMR signals to generate a
sine-modulated 2D NMR spectrum with an anti-phase peak pair derived
from said sine modulating wherein (1) the chemical shift value of
.sup.1H.sup..delta. is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup..delta.), and (2) the chemical shift
values of .sup.1H.sup..beta. and .sup.13C.sup..beta. are measured
in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming said anti-phase peak pair and the
frequency at the center of the two peaks, respectively, wherein
said sine-modulated 2D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sup..beta. in a phase sensitive
manner.
46. The method according to claim 45, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
value of .sup.13C.sup..beta. in a phase sensitive manner in
t.sub.1(.sup.13C.sup..beta.) and the chemical shift value of
.sup.1H.sup..delta. in t.sub.2(.sup.1H.sup..delta.) and (2) to
cosine modulate the .sup.13C.sup..beta. chemical shift evolution in
t.sub.1(.sup.13C.sup..beta.) with the chemical shift evolution of
.sup.1H.sup..beta. for the additional NMR signals, and said
processing the NMR signals and the additional NMR signals further
comprises generating a cosine-modulated 2D NMR spectrum with an
in-phase peak pair derived from said cosine modulating, a sum 2D
NMR spectrum generated by adding said sine-modulated 2D NMR
spectrum and said cosine-modulated 2D NMR spectrum, and a
difference 2D NMR spectrum generated by subtracting said
cosine-modulated 2D NMR spectrum from said sine-modulated 2D NMR
spectrum, wherein combined use of said sum 2D NMR spectrum and said
difference 2D NMR spectrum enables placement of the two peaks
forming said peak pairs into separate spectra, thereby allowing
phase-sensitive editing of the two peaks forming said peak
pairs.
47. The method according to claim 45, wherein said applying
radiofrequency pulses is carried out so that: (i) the chemical
shift evolution of a .delta.-carbon of an amino acid residue with
an aromatic side chain, .sup.13C.sup..delta., occurs under
conditions effective to generate NMR signals encoding the chemical
shift value of .sup.13C.sup..delta. in a phase sensitive manner in
an indirect time domain dimension, t.sub.3(.sup.13C.sup..delta.),
and said processing the NMR signals generates a three dimensional
(3D) NMR spectrum with a peak pair wherein (1) the chemical shift
values of .sup.1H.sup..delta. and .sup.13C.sup..delta. are measured
in two frequency domain dimensions,
.omega..sub.2(.sup.1H.sup..delta.) and
.omega..sub.3(.sup.13C.sup..delta.), respectively, and (2) the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming said peak pair and the frequency at
the center of the two peaks, respectively; or (ii) the chemical
shift evolution of a .gamma.-carbon of an amino acid residue with
an aromatic side chain, .sup.13C.sup..gamma. occurs under
conditions effective to generate NMR signals encoding the chemical
shift value of .sup.13C.sup..gamma., in a phase sensitive manner in
an indirect time domain dimension, t.sub.3(.sup.13C.sup..gamma.),
and said processing the NMR signals generates a three dimensional
(3D) NMR spectrum with a peak pair wherein (1) the chemical shift
values of .sup.1H.sup..delta. and .sup.13C.sup..gamma. are measured
in two frequency domain dimensions,
.omega..sub.2(.sup.1H.sup..delta.) and
.omega..sub.3(.sup.13C.sup..gamma.), respectively, and (2) the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming said peak pair and the frequency at
the center of the two peaks, respectively.
48. The method according to claim 46, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
value of .sup.13C.sup..beta. in a phase sensitive manner in
t.sub.1(.sup.13C.sup..beta.) and the chemical shift value of
.sup.1H.sup..delta. in t.sub.2(.sup.1H.sup..delta.), and (2) to
avoid sine modulating the chemical shift evolution of
.sup.13C.sup..beta. in t.sub.1(.sup.13C.sup..beta.) with the
chemical shift evolution of .sup.1H.sup..beta. for the additional
NMR signals, and said processing the NMR signals and the additional
NMR signals generates a 2D NMR spectrum with an additional peak
located centrally between said peak pair which measure the chemical
shift value of .sup.13C.sup..beta. along
.omega..sub.1(.sup.13C.sup..beta.).
49. The method according to claim 48, wherein said additional peak
is derived from .sup.13C.sup..beta. nuclear spin polarization.
50. The method according to claim 49, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2J, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup..beta. magnetization, and
.sup.1H.sup..beta. magnetization is transferred to
.sup.13C.sup..beta., to .sup.13C.sup..delta., and to
.sup.1H.sup..delta., where the NMR signal is detected.
51. The method according to claim 50, wherein said applying
radiofrequency pulses comprises: applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3J to
generate a first NMR signal, and applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3J,
wherein phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal, said method further
comprising: adding and subtracting the first NMR signal and the
second NMR signal prior to said processing, whereby said processing
the NMR signals generates a first NMR subspectrum derived from said
subtracting which contains said peak pair, and a second NMR
subspectrum derived from said adding which contains said additional
peak located centrally between the two peaks forming said peak
pair.
52. A method of conducting a reduced dimensionality two-dimensional
(2D) H,C,H-COSY nuclear magnetic resonance (NMR) experiment by
measuring the chemical shift values for .sup.1H.sup.m,
.sup.13C.sup.m, and .sup.1H.sup.n of a protein molecule wherein m
and n indicate atom numbers of two CH, CH.sub.2 or CH.sub.3 groups
in an amino acid residue, wherein the chemical shift values of
.sup.1H.sup.m and .sup.13C.sup.m which are encoded in a peak pair
of a 2D NMR spectrum are detected in a phase sensitive manner, said
method comprising: providing a protein sample; applying
radiofrequency pulses to the protein sample which effect a nuclear
spin polarization transfer wherein the chemical shift evolutions of
.sup.1H.sup.m and .sup.13C.sup.m are connected to the chemical
shift evolution of .sup.1H.sup.n, under conditions effective (1) to
generate NMR signals encoding the chemical shift value of
.sup.13C.sup.m in a phase sensitive manner in an indirect time
domain dimension, t.sub.1(.sup.13C.sup.m), and the chemical shift
value of .sup.1H.sup.n in a direct time domain dimension,
t.sub.2(.sup.1H.sup.n) and (2) to sine modulate the chemical shift
evolution of .sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the
chemical shift evolution of .sup.1H.sup.m; and processing the NMR
signals to generate a sine-modulated 2D NMR spectrum with
anti-phase peak pairs derived from said sine modulating wherein (1)
the chemical shift value of .sup.1H.sup.n is measured in a
frequency domain dimension, .omega..sub.2(.sup.1H.sup.n), and (2)
the chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.m), by the frequency differences between
each of the two peaks forming each of said anti-phase peak pairs
and the frequencies at the center of the two peaks, respectively,
wherein said sine-modulated 2D NMR spectrum enables detection of
the chemical shift value of .sup.1H.sup.m in a phase sensitive
manner.
53. The method according to claim 52, wherein said applying
radiofrequency pulses is carried out under conditions effective (1)
to generate additional NMR signals encoding the chemical shift
value of .sup.13C.sup.m in a phase sensitive manner in
t.sub.1(.sup.13C.sup.m) and the chemical shift value of
.sup.1H.sup.n in t.sub.2(.sup.13H.sup.n) and (2) to cosine modulate
the .sup.13C.sup.m chemical shift evolution in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sub.m for the additional NMR signals, and said processing
the NMR signals and the additional NMR signals further comprises
generating a cosine-modulated 2D NMR spectrum with in-phase peak
pairs derived from said cosine modulating, a sum 2D NMR spectrum
generated by adding said sine-modulated 2D NMR spectrum and said
cosine-modulated 2D NMR spectrum, and a difference 2D NMR spectrum
generated by subtracting said cosine-modulated 2D NMR spectrum from
said sine-modulated 2D NMR spectrum, wherein combined use of said
sum 2D NMR spectrum and said difference 2D NMR spectrum enables
placement of the two peaks forming said peak pairs into separate
spectra, thereby allowing phase-sensitive editing of the two peaks
forming said peak pairs.
54. The method according to claim 52, wherein said applying
radiofrequency pulses effects a nuclear spin polarization transfer
according to FIG. 2K, wherein a radiofrequency pulse is used to
create transverse .sup.1H.sup.m magnetization, and .sup.1H.sup.m
polarization is transferred to .sup.13C.sup.m, to .sup.1H.sup.m,
and to .sup.1H.sup.n, where the NMR signal is detected.
55. The method according to claim 54, wherein said applying
radiofrequency pulses is carried out according to the scheme shown
in FIG. 3K.
56. A method for sequentially assigning chemical shift values of an
.alpha.-proton, .sup.1H.sup..alpha., an .alpha.-carbon,
.sup.13C.sup..alpha., a polypeptide backbone amide nitrogen,
.sup.15N, and a polypeptide backbone amide proton, .sup.1H.sup.N,
of a protein molecule comprising: providing a protein sample;
conducting a set of reduced dimensionality (RD) nuclear magnetic
resonance (NMR) experiments on the protein sample, wherein the
chemical shift values of .sup.1H.sup..alpha. and
.sup.13C.sup..alpha. which are encoded in a peak pair of a 3D NMR
spectrum are detected in a phase sensitive manner, comprising: (1)
a RD three dimensional (3D) HA,CA,(CO),N,HN NMR experiment to
measure and connect chemical shift values of the .alpha.-proton of
amino acid residue i-1, .sup.1H.sup..alpha..sub.i-1, the
.alpha.-carbon of amino acid residue i-1,
.sup.13C.sup..alpha..sub.i-1, the polypeptide backbone amide
nitrogen of amino acid residue i, .sup.15N.sub.i, and the
polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i and (2) a RD 3D HNNCAHA NMR experiment to
measure and connect the chemical shift values of the .alpha.-proton
of amino acid residue i, .sup.1H.sup..alpha..sub.i, the
.alpha.-carbon of amino acid residue i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i; and obtaining sequential
assignments of the chemical shift values of .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N by (i) matching
the chemical shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 with the chemical shift values of
.sup.1H.sup..alpha..sub.i and .sup.13C.sup..alpha..sub.i, (ii)
using the chemical shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 to identify the type of amino acid
residue i-1, and (iii) mapping sets of sequentially connected
chemical shift values to the amino acid sequence of the polypeptide
chain and using said chemical shift values to locate secondary
structure elements within the polypeptide chain.
57. The method according to claim 56 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NUN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i-1, .sup.1H.sup..beta..sub.i-1, the
.beta.-carbon of amino acid residue i-1,
.sup.13C.sup..beta..sub.i-1, .sup.1H.sup..alpha..sub.i-1,
.sup.13C.sup..alpha..sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
by using the chemical shift values of .sup.1H.sup..beta..sub.i-1
and .sup.13C.sup..beta..sub.i-1 to identify the type of amino acid
residue i-1.
58. The method according to claim 57 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by said RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by said
RD 3D HA,CA,(CO),N,HN NMR experiment, RD 3D HNNCAHA NMR experiment,
and RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment.
59. The method according to claim 57 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i; and obtaining sequential
assignments by matching the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i with the
chemical shift values of .sup.1H.sup..beta..sub.i-1,
.sup.13C.sup..beta..sub.i-1, .sup.1H.sup..alpha..sub.i-1, and
.sup.13C.sup..alpha..sub.i-1 measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
60. The method according to claim 57 further comprising: subjecting
the protein sample to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift value of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measure by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
61. The method according to claim 57 further comprising: subjecting
the protein sample to a RD two-dimensional (2D) HB,CB,(CG,CD),HD
NMR experiment to measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1, and a
.delta.-proton of amino acid residue i-1 with an aromatic side
chain, .sup.1H.sup..delta..sub.i-1; and obtaining sequential
assignments by (i) matching the chemical shift values of
.sup.1H.sup..beta..sub.i-1 and .sup.13C.sup..beta..sub.i-1 measured
by said RD 2D HB, CB, (CG,CD), HD NMR experiment with the chemical
shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta. measured
by said RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, (ii) using said chemical shift values to identify amino
acid residue i as having an aromatic side chain, and (iii) mapping
sets of sequentially connected chemical shift values to the amino
acid sequence of the polypeptide chain and locating amino acid
residues with aromatic side chains along said polypeptide
chain.
62. The method according to claim 57 further comprising: subjecting
the protein sample to a RD 3D H,C,C,H-COSY NMR experiment or a RD
3D H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons of amino acid residue i,
.sup.1H.sup.ali.sub.i, and aliphatic carbons of amino acid residue
i, .sup.13C.sup.ali.sub.i; and obtaining sequential assignments of
the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i, by (i) matching the chemical shift values
of .sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i measured
using said RD 3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY
RD NMR experiment with the chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha..sub.i,
and .sup.13C.sup..alpha..sub.i measured by said RD 3D
HA,CA,(CO),N,HN NMR experiment, RD 3D HNNCAHA NMR experiment, and
RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and (ii) using the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, to identify the
type of amino acid residue i.
63. The method according to claim 56 further comprising: subjecting
the protein sample to a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of a polypeptide
backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i-1 by matching the chemical
shift value of .sup.13C.sup..alpha..sub.i measured by said RD 3D
HNN<CO,CA> NMR experiment with the sequentially assigned
chemical shift values of .sup.13C.sup..alpha., .sup.15N, and
.sup.1H.sup.N measured by said RD 3D HA,CA,(CO),N,HN NMR experiment
and RD 3D HNNCAHA NMR experiment.
64. The method according to claim 56 further comprising: subjecting
the protein sample to (i) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
the .alpha.-proton of amino acid residue i,
.sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid residue
i, .sup.13C.sup..alpha..sub.i, and a polypeptide backbone carbonyl
carbon of amino acid residue i, .sup.13C'.sub.i and (ii) a RD 3D
HNN<CO,CA> NMR experiment to measure and connect the chemical
shift values of .sup.13C'.sub.i, the .alpha.-carbon of amino acid
residue i+1, .sup.13C.sup..alpha..sub.i+1, the polypeptide backbone
amide nitrogen of amino acid residue i+1, .sup.15N.sub.i+1, and the
polypeptide backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1; and obtaining sequential assignments by
matching the chemical shift value of .sup.13C'.sub.i measured by
said RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment.
65. The method according to claim 56, further comprising:
subjecting the protein sample to a RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment to measure and connect the chemical shift values of
aliphatic protons of amino acid residue i-1,
.sup.1H.sup.ali.sub.i-1, aliphatic carbons of amino acid residue
i-1, .sup.13C.sup.ali.sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 for amino acid residues i having unique
pairs of .sup.15N.sub.i and .sup.1H.sup.N.sub.i chemical shift
values by matching the chemical shift values of .sup.1H.sup..alpha.
and .sup.13C.sup..alpha. measured by said RD 3D HNNCAHA NMR
experiment and RD 3D HA,CA,(CO),N,HN NMR experiment with the
chemical shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 measured by said RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment and using the
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 chemical shift
values to identify the type of amino acid residue i-1.
66. The method according to claim 56 further comprising: subjecting
the protein sample to a RD 3D H,C,C,H-COSY NMR experiment or a RD
3D H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons of amino acid residue i,
.sup.1H.sup.ali.sub.i, and aliphatic carbons of amino acid residue
i, .sup.13C.sup.ali.sub.i; and obtaining sequential assignments of
the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i by (i) matching the chemical shift values of
.sup.1H.sup..alpha..sub.i and .sup.13C.sup..alpha..sub.i measured
using said RD 3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY
RD NMR experiment with the chemical shift values of
.sup.1H.sup..alpha. and .sup.13C.sup..alpha. measured by said RD 3D
HA,CA,(CO),N,HN NMR experiment and RD 3D HNNCAHA NMR experiment and
(ii) using the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i, to identify the type of amino acid residue
i.
67. A method for sequentially assigning chemical shift values of a
.beta.-proton, .sup.1H.sup..beta., a .beta.-carbon,
.sup.13C.sup..beta., an .alpha.-proton, .sup.1H.sup..alpha., an
.alpha.-carbon, .sup.13C.sup..alpha., a polypeptide backbone amide
nitrogen, .sup.15N, and a polypeptide backbone amide proton,
.sup.1H.sup.N, of a protein molecule comprising: providing a
protein sample; conducting a set of reduced dimensionality (RD)
nuclear magnetic resonance (NMR) experiments on the protein sample,
wherein the chemical shift values of .sup.1H.sup..alpha./.beta. and
.sup.13C.sup..alpha./.beta. which are encoded in peak pairs of a 3D
NMR spectrum are detected in a phase sensitive manner, comprising:
(1) a RD three-dimensional (3D)
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i-1, .sup.1H.sup..beta..sub.1-i, the
.beta.-carbon of amino acid residue i-1,
.sup.13C.sup..beta..sub.i-1, the .alpha.-proton of amino acid
residue i-1, .sup.1H.sup..alpha..sub.i-1, the .alpha.-carbon of
amino acid residue i-1, .sup.13C.sup..alpha..sub.i-1, the
polypeptide backbone amide nitrogen of amino acid residue i,
.sup.15N.sub.i, and the polypeptide backbone amide proton of amino
acid residue i, .sup.1H.sup.N.sub.i and (2) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
the .alpha.-proton of amino acid residue i,
.sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid residue
i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift values of .sup.1H.sup..beta., .sup.13C.sup..beta.,
.sup.1H.sup..alpha., .sup.13C.sup..alpha., .sup.15N, and
.sup.1H.sup.N by (i) matching the chemical shift values of the
.alpha.- and .beta.-protons of amino acid residue i-1,
.sup.1H.sup..alpha./.beta..sub.i-1, and the .alpha.- and
.beta.-carbons of amino acid residue i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, with the chemical shift values
of .sup.1H.sup..alpha./.beta..sub.i and
.sup.13C.sup..alpha./.beta..sub.i, (ii) using the chemical shift
values of .sup.1H.sup..alpha./.beta..sub.i-1 and
.sup.13C.sup..alpha./.beta..sub.i, to identify the type of amino
acid residue i-1, and (iii) mapping sets of sequentially connected
chemical shift values to the amino acid sequence of the polypeptide
chain and using said chemical shift values to locate secondary
structure elements within the polypeptide chain.
68. The method according to claim 67 further comprising: subjecting
the protein sample to a RD 3D HA,CA,(CO),N,HN NMR experiment (i) to
measure and connect chemical shift values of
.sup.1H.sup..alpha..sub.i-1, .sup.13C.sup..alpha..sub.i-1,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i and (ii) to distinguish
between NMR signals for .sup.1H.sup..alpha./.sup.13C.sup..alpha.
and .sup.1H.sup..beta./.sup.13C.sup..beta. measured in said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.N,HN NMR
experiment.
69. The method according to claim 67 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by said RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by said
RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN
NMR experiment.
70. The method according to claim 67 further comprising: subjecting
the protein sample to a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of a polypeptide
backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i-1, .sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i,
and .sup.1H.sup.N.sub.i; and obtaining sequential assignments of
the chemical shift value of .sup.13C'.sub.i-1 by matching the
chemical shift value of .sup.13C.sup..alpha..sub.i measured by said
RD 3D HNN<CO,CA> NMR experiment with the sequentially
assigned chemical shift values of .sup.13C.sup..alpha., .sup.15N,
and .sup.1H.sup.N measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR
experiment.
71. The method according to claim 67 further comprising: subjecting
the protein sample to (i) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i and (ii) a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of .sup.13C'.sub.i,
the .alpha.-carbon of amino acid residue i+1,
.sup.13C.sup..alpha..sub.i+1, the polypeptide backbone amide
nitrogen of amino acid residue i+1, .sup.15N.sub.i+1, and the
polypeptide backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1; and obtaining sequential assignments by
matching the chemical shift value of .sup.13C'.sub.i measured by
said RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by said RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment.
72. The method according to claim 67 further comprising: subjecting
the protein sample to a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment
to measure and connect the chemical shift values of
.sup.1H.sup.ali.sub.i-1, .sup.13C.sup.ali.sub.i-1, .sup.15N.sub.i,
and .sup.1H.sup.N.sub.i; and obtaining sequential assignments of
the chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 for amino acid residues i having unique
pairs of .sup.15N.sub.i and .sup.1H.sup.N.sub.i chemical shift
values by matching the chemical shift values of .sup.1H.sup..beta.,
.sup.13C.sup..beta..sub., .sup.1H.sup..alpha., and
.sup.13C.sup..alpha. measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment
with the chemical shift values of .sup.1H.sup..beta..sub.i-1,
.sup.13C.sup..beta..sub.i-1, .sup.1H.sup..alpha..sub.i-1, and
.sup.13C.sup..alpha..sub.i-1 measured by said RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment and using the
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 chemical shift
values to identify the type of amino acid residue i-1.
73. The method according to claim 67 further comprising: subjecting
the protein sample to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift value of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measured by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
74. The method according to claim 67 further comprising: subjecting
the protein sample to a RD two-dimensional (2D) HB,CB,(CG,CD),HD
NMR experiment to measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i, and a
.delta.-proton of amino acid residue i with an aromatic side chain,
.sup.1H.sup..delta..sub.i; and obtaining sequential assignments by
(i) matching the chemical shift values of .sup.1H.sup..beta..sub.i
and .sup.13C.sup..beta..sub.i measured by said RD 2D HC,CB, (CG,
CD), HDNMR experiment with the chemical shift values of
.sup.1H.sup..beta. and .sup.13C.sup..beta. measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment,
(ii) using said chemical shift values to identify amino acid
residue i as having an aromatic side chain, and (iii) mapping sets
of sequentially connected chemical shift values to the amino acid
sequence of the polypeptide chain and locating amino acid residues
with aromatic side chains along said polypeptide chain.
75. The method according to claim 67, further comprising:
subjecting the protein sample to a RD 3D H,C,C,H-COSY NMR
experiment or a RD 3D H,C,C,H-TOCSY NMR experiment to measure and
connect the chemical shift values of aliphatic protons of amino
acid residue i, .sup.1H.sup.ali i and aliphatic carbons of amino
acid residue i, .sup.13C.sup.ali i; and obtaining sequential
assignments of the chemical shift values of .sup.1H.sup.ali.sub.i
and .sup.13C.sup.ali.sub.i by (i) matching the chemical shift
values of .sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i measured
using said RD 3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY
RD NMR experiment with the chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha., and
.sup.13C.sup..alpha. measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment,
and (ii) using the chemical shift values of .sup.1H.sup.ali.sub.i
and .sup.13C.sup.ali.sub.i, to identify the type of amino acid
residue i.
76. A method for sequentially assigning the chemical shift values
of aliphatic protons, .sup.1H.sup.ali, aliphatic carbons,
.sup.13C.sup.ali, a polypeptide backbone amide nitrogen, .sup.15N,
and a polypeptide backbone amide proton, .sup.1H.sup.N, of a
protein molecule comprising: providing a protein sample; conducting
a set of reduced dimensionality (RD) nuclear magnetic resonance
(NMR) experiments on the protein sample, wherein the chemical shift
values of .sup.1H.sup.ali and .sup.13C.sup.ali which are encoded in
peak pairs of a 3D NMR spectrum are detected in a phase sensitive
manner, comprising: (1) a RD three-dimensional (3D)
H,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the
chemical shift values of the aliphatic protons of amino acid
residue i-1, .sup.1H.sup.ali.sub.i-1, the aliphatic carbons of
amino acid residue i-1, .sup.13C.sup.ali.sub.i-1, the polypeptide
backbone amide nitrogen of amino acid residue i, .sup.15N.sub.i,
and the polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i and (2) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
the .alpha.-proton of amino acid residue i,
.sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid residue
i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift values of .sup.1H.sup.ali, .sup.13C.sup.ali,
.sup.15N, and .sup.1H.sup.N by (i) matching the chemical shift
values of the .alpha.- and .beta.-protons of amino acid residue
i-1, .sup.1H.sup..alpha./.beta..sub.i-1 and the .alpha.- and
.beta.-carbons of amino acid residue i-1,
.sup.13C.sup..alpha./.beta..sub.i-1 with the chemical shift values
of .sup.1H.sup..alpha./.beta..sub.i and
.sup.13C.sup..alpha./.beta..sub.i of amino acid residue i, (ii)
using the chemical shift values of .sup.1H.sup.ali.sub.i-i and
.sup.13C.sup.ali.sub.i-1 to identify the type of amino acid residue
i-1, and (iii) mapping sets of sequentially connected chemical
shift values to the amino acid sequence of the polypeptide chain
and using said chemical shift values to locate secondary structure
elements within the polypeptide chain.
77. The method according to claim 76 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta., CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by said
RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment.
78. The method according to claim 76 further comprising: subjecting
the protein sample to a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of a polypeptide
backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i-1 by matching the chemical
shift value of .sup.13C.sup..alpha..sub.i measured by said RD 3D
HNN<CO,CA> NMR experiment with the sequentially assigned
chemical shift values of .sup.13C.sup..alpha., .sup.15N, and
.sup.1H.sup.N measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment and RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN
NMR experiment.
79. The method according to claim 76 further comprising: subjecting
the protein sample to (i) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i and (ii) a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of .sup.13C'.sub.i,
the .alpha.-carbon of amino acid residue i+1,
.sup.13C.sup..alpha..sub.i+1, the polypeptide backbone amide
nitrogen of amino acid residue i+1, .sup.15N.sub.i+1, and the
polypeptide backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1; and obtaining sequential assignments by
matching the chemical shift value of .sup.13C'.sub.i measured by
said RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment.
80. The method according to claim 76 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment (i)
to measure and connect the chemical shift values of
.sup.1H.sup..alpha./.beta..sub.i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i, and (ii) to identify NMR signals for
.sup.1H.sup..alpha./.beta..sub.i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i in said RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment.
81. The method according to claim 76 further comprising: subjecting
the protein sample to a RD 3D HA,CA,(CO),N,HN NMR experiment (i) to
measure and connect chemical shift values of
.sup.1H.sup..alpha..sub.i-1, .sup.13C.sup..alpha..sub.i-1,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i and (ii) to identify NMR
signals for .sup.1H.sup..alpha. and .sup.13C.sup..alpha. in said RD
3D H,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment.
82. The method according to claim 76 further comprising: subjecting
the protein sample to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift value of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measured by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
83. The method according to claim 76 further comprising: subjecting
the protein sample to a RD two-dimensional (2D) HB,CB,(CG,CD),HD
NMR experiment to measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i, and a
.delta.-proton of amino acid residue i with an aromatic side chain,
.sup.1H.sup..delta..sub.i; and obtaining sequential assignments by
matching the chemical shift values of .sup.1H.sup..beta..sub.i and
.sup.13C.sup..beta..sub.i measured by said RD 2D HB,CB, (CG, CD),
HD NMR experiment with the chemical shift values of
.sup.1H.sup..beta. and .sup.13C.sup..beta. measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment and RD
3D H,C,(C-TOCSY-CO),N,HN NMR experiment, using said chemical shift
values to identify amino acid residue i as having an aromatic side
chain, and mapping sets of sequentially connected chemical shift
values to the amino acid sequence of the polypeptide chain and
locating amino acid residues with aromatic side chains along said
polypeptide chain.
84. The method according to claim 76 further comprising: subjecting
the protein sample to a RD 3D H,C,C,H-COSY NMR experiment or a RD
3D H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons of amino acid residue i,
.sup.1H.sup.ali.sub.i and aliphatic carbons of amino acid residue
i, .sup.13C.sup.ali.sub.i; and obtaining sequential assignments of
the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i by (i) matching the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i measured using
said RD 3D H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY NMR
experiment with the chemical shift values of .sup.1H.sup.ali and
.sup.13C.sup.ali measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment and RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN
NMR experiment, and (ii) using the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, to identify the
type of amino acid residue i.
85. A method for sequentially assigning chemical shift values of
aliphatic protons, .sup.1H.sup.ali, aliphatic carbons,
.sup.13C.sup.ali, a polypeptide backbone amide nitrogen, .sup.15N,
and a polypeptide backbone amide proton, .sup.1H.sup.N, of a
protein molecule comprising: providing a protein sample; conducting
a set of reduced dimensionality (RD) nuclear magnetic resonance
(NMR) experiments on the protein sample, wherein the chemical shift
values of .sup.1H.sup.ali and .sup.13C.sup.ali which are encoded in
peak pairs of a 3D NMR spectrum are detected in a phase sensitive
manner, comprising: (1) a RD three-dimensional (3D)
H,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the
chemical shift values of the aliphatic protons of amino acid
residue i-1, .sup.1H.sup.ali.sub.i-1, the aliphatic carbons of
amino acid residue i-1, .sup.13C.sup.ali.sub.i-1, the polypeptide
backbone amide nitrogen of amino acid residue i, .sup.15N.sub.i,
and the polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i and (2) a RD 3D HNNCAHA NMR experiment to
measure and connect the chemical shift values of the .alpha.-proton
of amino acid residue i, .sup.1H.sup..alpha..sub.i, the
.alpha.-carbon of amino acid residue i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i; and obtaining sequential
assignments of the chemical shift values of .sup.1H.sup.ali,
.sup.13C.sup.ali, .sup.15N, and .sup.1H.sup.N by (i) matching the
chemical shift values of the .alpha.-proton of amino acid residue
i-1, .sup.1H.sup..alpha..sub.i-1, and the .alpha.-carbon of amino
acid residue i-1, .sup.13C.sup..alpha..sub.i-1, with the chemical
shift values of .sup.1H.sup..alpha..sub.i and
.sup.13C.sup..alpha..sub.i, (ii) using the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 to identify
the type of amino acid residue i-1, and (iii) mapping sets of
sequentially connected chemical shift values to the amino acid
sequence of the polypeptide chain and using said chemical shift
values to locate secondary structure elements within the
polypeptide chain.
86. The method according to claim 85 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta., CO,HA NMR experiment to
measure and connect the chemical shift values of a .beta.-proton of
amino acid residue i, .sup.1H.sup..beta..sub.i, a .beta.-carbon of
amino acid residue i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..beta., .sup.15N, and .sup.1H.sup.N measured by said
RD 3D H C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNNCAHA NMR
experiment.
87. The method according to claim 85 further comprising: subjecting
the protein sample to a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of a polypeptide
backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments of the
chemical shift value of .sup.13C'.sub.i-1 by matching the chemical
shift value of .sup.13C.sup..alpha..sub.i measured by said RD 3D
HNN<CO,CA> NMR experiment with the sequentially assigned
chemical shift values of .sup.13C.sup..alpha., .sup.15N, and
.sup.1H.sup.N measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment and RD 3D HNNCAHA NMR experiment.
88. The method according to claim 85 further comprising: subjecting
the protein sample to (i) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of a .beta.-proton of
amino acid residue i, .sup.1H.sup..beta..sub.i, a .beta.-carbon of
amino acid residue i, .sup.13C.sup..beta..sub.i, the .alpha.-proton
of amino acid residue i, .sup.1H.sup..alpha..sub.i, the
.alpha.-carbon of amino acid residue i, .sup.13C.alpha..sub.i, and
a polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i and (ii) a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of .sup.13C'.sub.i,
an .alpha.-carbon of amino acid residue i+1,
.sup.13C.sup..alpha..sub.i+1, a polypeptide backbone amide nitrogen
of amino acid residue i+1, .sup.15N.sub.i+1, and a polypeptide
backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1; and obtaining sequential assignments by
matching the chemical shift value of .sup.13C'.sub.i measured by
said RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by said RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment.
89. The method according to claim 85 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NUN NMR experiment (i)
to measure and connect the chemical shift values of the .alpha.-
and .beta.-protons of amino acid residue i-1,
.sup.1H.sup..alpha./.beta..sub.i-1, .alpha.- and .beta.-carbons of
amino acid residue i-1, .sup.13C.sup..alpha./.beta..sub.i-1,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i, and (ii) to distinguish
NMR signals for the chemical shift values of
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1,
.sup.1H.sup..alpha..sub.i-1, and .sup.13C.sup..alpha..sub.i-1
measured by said RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment from
NMR signals for the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 other than
.sup.1H.sup..alpha./.beta..sub.i-1 and
.sup.13C.sup..alpha./.beta..sub.i-1 measured by said RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment.
90. The method according to claim 85 further comprising: subjecting
the protein sample to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i; and obtaining sequential
assignments by matching the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i measured
by said RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR
experiment with the chemical shift values of
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1,
.sup.1H.sup..alpha..sub.i-1, and .sup.13C.sup..alpha..sub.i-1
measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
91. The method according to claim 85 further comprising: subjecting
the protein sample to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift values of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i; and obtaining sequential assignments by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measured by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by said RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
92. The method according to claim 85 further comprising: subjecting
the protein sample to a RD two-dimensional (2D) HB,CB,(CG,CD),HD
NMR experiment to measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i, and a
.delta.-proton of amino acid residue i with an aromatic side chain,
.sup.1H.sup..delta..sub.i; and obtaining sequential assignments by
matching the chemical shift values of .sup.1H.sup..beta..sub.i and
.sup.13C.sup..beta..sub.i measured by said RD 2D HB, CB,(CG, CD),
HD NMR experiment with the chemical shift values of
.sup.1H.sup..beta. and .sup.13C.sup..beta. measured by said RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment, using said chemical shift
values to identify amino acid residue i as having an aromatic side
chain, and mapping sets of sequentially connected chemical shift
values to the amino acid sequence of the polypeptide chain and
locating amino acid residues with aromatic side chains along said
polypeptide chain.
93. The method according to claim 85 further comprising: subjecting
the protein sample to a RD 3D H,C,C,H-COSY NMR experiment or a RD
3D H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons of amino acid residue i,
.sup.1H.sup.ali.sub.i and aliphatic carbons of amino acid residue
i, .sup.13C.sup.ali.sub.i; and obtaining sequential assignments of
the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i by (i) matching the chemical shift values of
.sup.1H.sup.ali and .sup.13C.sup.ali measured using said RD 3D
H,C,C,H-COSY NMR experiment or RD 3D H,C,C,H-TOCSY NMR experiment
with the chemical shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by said RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment and RD 3D HNNCAHA NMR
experiment, and (ii) using the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, to identify the
type of amino acid residue i.
Description
[0001] The present invention is a divisional of U.S. patent
application Ser. No. 10/628,818, filed Jul. 28, 2003, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/399,332, filed Jul. 26, 2002, which is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of using phase
sensitively-detected reduced dimensionality nuclear magnetic
resonance (NMR) spectroscopy for obtaining chemical shift
assignment and for structure determination of proteins.
BACKGROUND OF THE INVENTION
[0004] The use of triple resonance (TR) nuclear magnetic resonance
(NMR) experiments for the resonance assignment of polypeptide
chains via heteronuclear scalar connectivities (Montelione et al.,
J. Am. Chem. Soc., 111:5474-5475 (1989); Montelione et al., J.
Magn. Reson., 87:183-188 (1989); Kay et al., J. Magn. Reson.,
89:496-514 (1990); Ikura et al., Biochemistry, 29:4659-8979 (1990);
Edison et al., Methods Enzymol., 239:3-79 (1994)) is a standard
approach which neatly complements the assignment protocol based on
.sup.1H--.sup.1H nuclear Overhauser effects (NOE) (Wuthrich, NMR of
Proteins and Nucleic Acids, Wiley, New York (1986)). In addition,
triple resonance NMR spectra are highly amenable to a fast
automated analysis (Friedrichs et al., J. Biomol. NMR, 4:703-726
(1994); Zimmerman et al., J. Biomol. NMR, 4:241-256 (1994); Bartels
et al., J. Biomol. NMR, 7:207-213 (1996); Morelle et al., J.
Biomol. NMR, 5:154-160 (1995); Buchler et al., J. Magn. Reson.,
125:34-42 (1997); Lukin et al., J. Biomol. NMR, 9:151-166 (1997)),
yielding the .sup.13C.sup..alpha./.beta. chemical shifts at an
early stage of the assignment procedure. This enables both the
identification of regular secondary structure elements without
reference to NOEs (Spera et al., J. Am. Chem. Soc., 113: 5490-5491
(1991)) and the derivation of (.phi.,.psi.)-angle constraints which
serve to reduce the number of cycles consisting of nuclear
Overhauser enhancement spectroscopy (NOESY) peak assignment and
structure calculation (Luginbuhl et al., J. Magn. Reson., B
109:229-233 (1995)).
[0005] NMR assignments are prerequisite for NMR-based structural
biology (Wuthrich, NMR of Proteins and Nucleic Acid, Wiley:New York
(1986)) and, thus, for highthroughput (HTP) structure determination
in structural genomics (Rost, Structure, 6:259-263 (1998);
Montelione et al., Nature Struct. Biol., 6:11-12. (1999); Burley,
Nature Struc Biol., 7:932-934 (2000)) and for exploring
structure-activity relationships (SAR) by NMR for drug discovery
(Shuker et al., Science, 274:1531-1534 (1996)). The aims of
structural genomics are to (i) explore the naturally occurring
"protein fold space" and (ii) contribute to the characterization of
function through the assignment of atomic resolution
three-dimensional (3D) structures to proteins. It is now generally
acknowledged that NMR will play an important role in structural
genomics (Montelione et al., Nature Struc. Biol., 7:982-984
(2000)). The resulting demand for HTP structure determination
requires fast and automated NMR data collection and analysis
protocols (Moseley et al., Curr. Opin. Struct. Biol., 9:635-642
(1999)).
[0006] The establishment of a HTP NMR structural genomics pipeline
requires two key objectives in data collection. Firstly, the
measurement time should be minimized in order to (i) lower the cost
per structure and (ii) relax the constraint that NMR samples need
to be stable over a long period of measurement time. The recent
introduction of commercial cryogenic probes (Styles et al., J.
Magn. Reson., 60:397-404 (1984); Flynn et al., J. Am. Chem. Soc.,
122:4823-4824 (2000)) promises to reduce measurement times by about
a factor of ten or more, and will greatly impact the realization of
this first objective. Secondly, reliable automated spectral
analysis requires recording of a "redundant" set of
multidimensional NMR experiments each affording good resolution
(which requires appropriately long maximal evolution times in all
indirect dimensions). Concomitantly, it is desirable to keep the
total number of NMR spectra small in order to minimize
"interspectral" variations of chemical shift measurements, which
may impede automated spectral analysis. Straightforward
consideration of this second objective would suggest increasing the
dimensionality of the spectra, preferably by implementing a suite
of four- or even higher-dimensional NMR experiments. Importantly,
however, the joint realization of the first and second objectives
is tightly limited by the rather large lower bounds of
higher-dimensional TR NMR measurement times if appropriately long
maximal evolution times are chosen.
[0007] Hence, "sampling limited" and "sensitivity limited" data
collection regimes are distinguished, depending on whether the
sampling of the indirect dimensions or the sensitivity of the
multidimensional NMR experiments "per se" determines the minimally
achievable measurement time. As a matter of fact, the ever
increasing performance of NMR spectrometers will soon lead to the
situation where, for many protein samples, the sensitivity of the
NMR spectrometers do not constitute the prime bottleneck
determining minimal measurement times. Instead, the minimal
measurement times encountered for recording conventional
higher-dimensional NMR schemes will be "sampling limited,"
particularly as high sensitivity cryoprobes become generally
available. As structure determinations of proteins rely on nearly
complete assignment of chemical shifts, which are obtained using
multidimensional .sup.13C, .sup.15N, .sup.1H-TR NMR experiments
(Montelione et al., J. Am. Chem. Soc., 111:5474-5475 (1989);
Montelione, et al., J. Magn. Reson., 87:183-188 (1989); Ikura et
al., Biochemistry, 29:4659-8979 (1990)), the development of TR NMR
techniques that avoid the sampling limited regime represents a key
challenge for future biomolecular NMR methods development.
[0008] Reduced dimensionality (RD) TR NMR experiments (Szyperski et
al., J. Biomol. NMR, 3:127-132 (1993); Szyperski et al., J. Am.
Chem. Soc., 115:9307-9308 (1993); Szyperski et al., J. Magn.
Reson., B 105:188-191 (1994); Brutscher et al., J. Magn. Reson., B
105:77-82 (1994); Szyperski et al., J. Magn. Reson., B 108: 197-203
(1995); Brutscher et al., J. Biomol. NMR, 5:202-206 (1995); Lohr et
al., J. Biomol. NMR, 6:189-197 (1995); Szyperski et al., J. Am.
Chem. Soc., 118:8146-8147 (1996); Szyperski et al., J. Magn.
Reson., 28:228-232 (1997); Bracken et al., J. Biomol. NMR, 9:94-100
(1997); Sklenar et al., J. Magn. Reson., 130:119-124 (1998);
Szyperski et al., J. Biomol. NMR, 11:387-405 (1998)), designed for
simultaneous frequency labeling of two spin types in a single
indirect dimension, offer a viable strategy to circumvent recording
NMR spectra in a sampling limited fashion. RD NMR is based on a
projection technique for reducing the spectral dimensionality of TR
experiments: the chemical shifts of the projected dimension give
rise to a cosine-modulation of the transfer amplitude, yielding
in-phase peak pairs encoding n chemical shifts in a n-1 dimensional
spectrum (Szyperski et al., J. Biomol. NMR, 3:127-132 (1993);
Szyperski et al., J. Am. Chem. Soc., 115:9307-9308 (1993)). As a
key result, this allows recording projected four-dimensional (4D)
NMR experiments with maximal evolution times typically achieved in
the corresponding conventional 3D NMR experiments (Szyperski et
al., J. Biomol. NMR, 3:127-132 (1993); Szyperski et al., J. Am.
Chem. Soc., 115:9307-9308 (1993); Szyperski et al., J. Magn. Reson.
B 105:188-191 (1994); Szyperski et al., J. Magn. Reson., B 108:
197-203 (1995); Szyperski et al., J. Am. Chem. Soc., 118:8146-8147
(1996); Szyperski et al., J. Magn. Reson., 28:228-232 (1997);
Bracken et al., J. Biomol. NMR, 9:94-100 (1997); Sklenar et al., J.
Magn. Reson., 130:119-124 (1998); Szyperski et al., J. Biomol. NMR,
11:387-405 (1998)). Furthermore, axial coherences, arising from
either incomplete insensitive nuclei enhanced by polarization
transfer (INEPT) or heteronuclear magnetization, can be observed as
peaks located at the center of the in-phase peak pairs (Szyperski
et al., J. Am. Chem. Soc., 118:8146-8147 (1996)). This allows both
the unambiguous assignment of multiple doublets with degenerate
chemical shifts in the other dimensions and the identification of
peak pairs by symmetrization of spectral strips about the position
of the central peak (Szyperski et al., J. Am. Chem. Soc.,
118:8146-8147 (1996); Szyperski et al., J. Biomol. NMR, 11:387-405
(1998)). Hence, observation of central peaks not only restores the
dispersion of the parent, higher-dimensional experiment, but also
provides access to reservoir of axial peak magnetization (Szyperski
et al., J. Am. Chem. Soc., 118:8146-8147 (1996)). Historically, RD
NMR experiments were first designed to simultaneously recruit both
.sup.1H and heteronuclear magnetization (Szyperski et al., J. Am.
Chem. Soc., 118:8146-8147 (1996)) for signal detection, a feature
that has also gained interest for improving transverse
relaxation-optimized spectroscopy (TROSY) pulse schemes (Pervushin
et al., Proc. Natl. Acad. Sci. USA, 94:12366-12371 (1997); Salzmann
et al., J. Am. Chem. Soc., 121:844-848 (1999); Pervushin et al., J.
Biomol. NMR, 12:345-348, (1998)). Moreover, RD two-spin coherence
NMR spectroscopy (Szyperski et al., J. Biomol. NMR, 3:127-132
(1993)) subsequently also called zero-quantum/double-quantum
(ZQ/DQ) NMR spectroscopy (Rexroth et al., J. Am. Chem. Soc.,
17:10389-10390 (1995)), served as a valuable radio-frequency (r.f.)
pulse module for measurement of scalar coupling constants (Rexroth
et al., J. Am. Chem. Soc., 17: 10389-10390 (1995)) and
cross-correlated heteronuclear relaxation (Reif et al., Science,
276:1230-1233 (1997); Yang et al., J. Am. Chem. Soc., 121:3555-3556
(1999); Chiarparin et al., J. Am. Chem. Soc., 122:1758-1761 (2000);
Brutscher et al., J. Magn. Reson., 130:346-351 (1998); Brutscher,
Concepts Magn. Reson., 122:207-229 (2000)).
[0009] Recently, a suite of RD .sup.13C,.sup.15N,.sup.1H-triple
resonance NMR experiments was presented for rapid and complete
protein resonance assignment (Szyperski et al., Proc. Natl. Acad.
Sci. USA, 99:8009-8014 (2002)). Even when using short measurement
times, these experiments allow one to retain the high spectral
resolution required for efficient automated analysis. "Sampling
limited" and "sensitivity limited" data collection regimes were
defined, respectively, depending on whether the sampling of the
indirect dimensions or the sensitivity of a multidimensional NMR
experiments per se determines the minimally required measurement
time. It was shown that RD NMR spectroscopy is a powerful approach
to avoid the "sampling limited regime", i.e., a standard set of
experiments allows one to effectively adapt minimal measurement
times to sensitivity requirements. It would be advantageous to
implement those RD NMR experiments in a manner that allows
frequency discrimination of the projected chemical shifts.
[0010] The present invention is directed to achieving these
objectives.
SUMMARY OF THE INVENTION
[0011] The present invention relates to a method of conducting a
reduced dimensionality three-dimensional (3D) HA,CA,(CO),N,HN
nuclear magnetic resonance (NMR) experiment by measuring the
chemical shift values for the following nuclei of a protein
molecule having two consecutive amino acid residues, i-1 and i: (1)
an .alpha.-proton of amino acid residue i-1,
.sup.1H.sup..alpha..sub.i-1; (2) an .alpha.-carbon of amino acid
residue i-1, .sup.13C.sup..alpha..sub.i; (3) a polypeptide backbone
amide nitrogen of amino acid residue i, .sup.15N.sub.i; and (4) a
polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i, where the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 which
are encoded in a peak pair of a 3D NMR spectrum are detected in a
phase sensitive manner. The method involves providing a protein
sample and applying radiofrequency pulses to the protein sample
which effect a nuclear spin polarization transfer where the
chemical shift evolutions of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1, of amino acid residue i-1 are
connected to the chemical shift evolutions of .sup.15N.sub.i and
.sup.1H.sup.N.sub.i of amino acid residue i, under conditions
effective (1) to generate NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i-1 and .sup.15N.sub.i in a
phase sensitive manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup..alpha.) and t.sub.2(.sup.15N), respectively,
and the chemical shift value of .sup.1H.sup.N.sub.i in a direct
time domain dimension, t.sub.3(.sup.1H.sup.N), and (2) to sine
modulate the .sup.13C.sup..alpha..sub.i-1 chemical shift evolution
in t.sub.1(.sup.13C.sup..alpha.) with the chemical shift evolution
of .sup.1H.sup..alpha..sub.i-1. Then, the NMR signals are processed
to generate a sine-modulated 3D NMR spectrum with an anti-phase
peak pair derived from the sine modulating, where (1) the chemical
shift values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i are measured
in two frequency domain dimensions, .omega..sub.2(.sup.15N) and
.omega..sub.3(.sup.1H.sup.N), respectively, and (2) the chemical
shift values of .sup.1H.sup.a.sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha.), by the frequency
difference between the two peaks forming the anti-phase peak pair
and the frequency at the center of the two peaks, respectively,
where the sine-modulated 3D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sup..alpha..sub.i-1 in a phase
sensitive manner.
[0012] The present invention also relates to a method of conducting
a reduced dimensionality three-dimensional (3D)
H,C,(C-TOCSY-CO),N,HN nuclear magnetic resonance (NMR) experiment
by measuring the chemical shift values for the following nuclei of
a protein molecule having two consecutive amino acid residues, i-1
and i: (1) aliphatic protons of amino acid residue i-1,
.sup.1H.sup.ali.sub.i-1; (2) aliphatic carbons of amino acid
residue i-1, .sup.13C.sup.ali.sub.i-1; (3) a polypeptide backbone
amide nitrogen of amino acid residue i, .sup.15N.sub.i; and (4) a
polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i, where the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 of amino acid residue i-1 are connected to
the chemical shift evolutions of .sup.15N.sub.i and
.sup.1H.sup.N.sub.i, of amino acid residue i, under conditions
effective (1) to generate a NMR signal encoding the chemical shifts
of .sup.13C.sup.ali.sub.i-1 and .sup.15N.sub.i in a phase sensitive
manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup.ali) and t.sub.2(.sup.15N), respectively, and
the chemical shift of .sup.1H.sup.N.sub.i in a direct time domain
dimension, t.sub.3(.sup.1H.sup.N), and (2) to sine modulate the
chemical shift evolutions of .sup.13C.sup.ali.sub.i-1 in
t.sub.1(.sup.13C.sup.ali) with the chemical shift evolutions of
.sup.1H.sup.ali.sub.i-1. Then, the NMR signals are processed to
generate a sine-modulated 3D NMR spectrum with anti-phase peak
pairs derived from the sine modulating where (1) the chemical shift
values of .sup.15 N.sub.i and .sup.1H.sup.N.sub.i are measured in
two frequency domain dimensions, .omega..sub.2(.sup.15N) and
.omega..sub.3(.sup.1H.sup.N), respectively, and (2) the chemical
shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.ali), by the frequency
differences between each of the two peaks forming each of the
anti-phase peak pairs and the frequencies at the center of the two
peaks, respectively, where the sine-modulated 3D NMR spectrum
enables detection of the chemical shift value of
.sup.1H.sup.ali.sub.i-1 in a phase sensitive manner.
[0013] Another aspect of the present invention relates to a method
of conducting a reduced dimensionality three-dimensional (3D)
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA nuclear magnetic
resonance (NMR) experiment by measuring the chemical shift values
for the following nuclei of a protein molecule having an amino acid
residue, i: (1) a .beta.-proton of amino acid residue i,
.sup.1H.sup..beta..sub.i; (2) a .beta.-carbon of amino acid residue
i, .sup.13C.sup..beta..sub.i; (3) an .alpha.-proton of amino acid
residue i, .sup.1H.sup..alpha..sub.i; (4) an .alpha.-carbon of
amino acid residue i, .sup.13C.sup..alpha..sub.i; and (5) a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i, where the chemical shift values of
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..alpha..sub.i, and
.sup.1H.sup..beta..sub.i/.sup.13C.sup..beta..sub.i which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup..alpha..sub.i,
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are connected to the chemical shift
evolution of .sup.13C'.sub.i, under conditions effective (1) to
generate NMR signals encoding the chemical shift values of
.sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.13C'.sub.i in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup..alpha./.beta.) and
t.sub.2(.sup.13C'), respectively, and the chemical shift value of
.sup.1H.sup..alpha..sub.i in a direct time domain dimension,
t.sub.3(.sup.1H.sup..alpha.), and (2) to sine modulate the chemical
shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..alpha..sub.i in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i, respectively. Then, the NMR signals are
processed to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift values of .sup.13C'.sub.i and
.sup.1H.sup..alpha..sub.i are measured in two frequency domain
dimensions, .omega..sub.2(.sup.13C') and
.omega..sub.3(.sup.1H.sup..alpha.), respectively, and (2) (i) the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequency differences between each of the two peaks forming each of
the anti-phase peak pairs, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming the anti-phase peak pairs,
where the sine-modulated 3D NMR spectrum enables detection of the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i in a phase sensitive manner.
[0014] A further aspect of the present invention relates to a
method of conducting a reduced dimensionality three-dimensional
(3D) H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN nuclear
magnetic resonance (NMR) experiment by measuring the chemical shift
values for the following nuclei of a protein molecule having an
amino acid residue, i: (1) a .beta.-proton of amino acid residue i,
.sup.1H.sup..beta..sub.i; (2) a .beta.-carbon of amino acid residue
i, .sup.13C.sup..beta..sub.i; (3) an .alpha.-proton of amino acid
residue i, .sup.1H.sup..alpha..sub.i; (4) an .alpha.-carbon of
amino acid residue i, .sup.13C.sup..alpha..sub.i; (5) a polypeptide
backbone amide nitrogen of amino acid residue i, .sup.15N.sub.i;
and (6) a polypeptide backbone amide proton of amino acid residue
i, .sup.1H.sup.N, where the chemical shift values of
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..alpha..sub.i and
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..beta..sub.i, which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup..alpha..sub.i,
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are connected to the chemical shift
evolutions of .sup.15N.sub.i and .sup.1H.sup.N.sub.i, under
conditions effective (1) to generate NMR signals encoding the
chemical shift values of .sup.13C.sup..alpha..sub.i,
.sup.13C.sup..beta..sub.i and .sup.15N.sub.i in a phase sensitive
manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i,
in a direct time domain dimension, t.sub.3(.sup.1H.sup.N), and (2)
to sine modulate the chemical shift evolutions of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i in
t.sub.1(.sup.13C.sup..alpha./.beta.) with the chemical shift
evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i, respectively. Then, the NMR signals are
processed to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i
are measured in two frequency domain dimensions,
.omega..sub.2(.sup.15N) and .omega..sub.3(.sup.1H.sup.N),
respectively, and (2) (i) the chemical shift values of
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i are measured
in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequency
differences between each of the two peaks forming each of the
anti-phase peak pairs, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..alpha..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming the anti-phase peak pairs,
where the sine-modulated 3D NMR spectrum enables detection of the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i in a phase sensitive manner.
[0015] The present invention also relates to a method of conducting
a reduced dimensionality three-dimensional (3D) H,C,C,H-COSY
nuclear magnetic resonance (NMR) experiment by measuring the
chemical shift values for .sup.1H.sup.m, .sup.13C.sup.m,
.sup.1H.sup.n, and .sup.13C.sup.n of a protein molecule where m and
n indicate atom numbers of two CH, CH.sub.2 or CH.sub.3 groups that
are linked by a single covalent carbon-carbon bond in an amino acid
residue, where the chemical shift values of .sup.1H.sup.m and
.sup.13C.sup.m, which are encoded in a peak pair of a 3D NMR
spectrum are detected in a phase sensitive manner. The method
involves providing a protein sample and applying radiofrequency
pulses to the protein sample which effects a nuclear spin
polarization transfer where the chemical shift evolutions of
.sup.1H.sup.m and .sup.13C.sup.m are connected to the chemical
shift evolutions of .sup.1H.sup.n and .sup.13C.sup.n, under
conditions effective (1) to generate NMR signals encoding the
chemical shift values of .sup.13C.sup.m and .sup.13C.sup.n, in a
phase sensitive manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n), respectively,
and the chemical shift value of .sup.1H.sup.n in a direct time
domain dimension, t.sub.3(.sup.1H.sup.n), and (2) to sine modulate
the chemical shift evolution of .sup.13C.sup.m in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sub.m. Then, the NMR signals are processed to generate a
sine-modulated 3D NMR spectrum with anti-phase peak pairs derived
from the sine modulating where (1) the chemical shift values of
.sup.13C.sup.n and .sup.1H.sup.n are measured in two frequency
domain dimensions, .omega..sub.2(.sup.13C.sup.n) and
.omega..sub.3(.sup.1H.sup.n), respectively, and (2) the chemical
shift values of .sup.1H.sup.m and .sup.13C.sup.m are measured in a
frequency domain dimension, .omega..sub.1(.sup.13C.sup.m), by the
frequency differences between each of the two peaks forming each of
the anti-phase peak pairs and the frequencies at the center of the
two peaks, respectively, where the sine-modulated 3D NMR spectrum
enables detection of the chemical shift value of .sup.1H.sub.m in a
phase sensitive manner.
[0016] Another aspect of the present invention relates to a method
of conducting a reduced dimensionality three-dimensional (3D)
H,C,C,H-TOCSY nuclear magnetic resonance (NMR) experiment by
measuring the chemical shift values for .sup.1H.sup.m,
.sup.13C.sup.m, .sup.1H.sup.n, and .sup.13C.sup.n of a protein
molecule where m and n indicate atom numbers of two CH, CH.sub.2 or
CH.sub.3 groups that may or may not be directly linked by a single
covalent carbon-carbon bond in an amino acid residue, where the
chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m which are
encoded in a peak pair of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup.m and .sup.13C.sup.m are connected
to the chemical shift evolutions of .sup.1H.sup.n and
.sup.13C.sup.n, under conditions effective (1) to generate NMR
signals encoding the chemical shift values of .sup.13C.sup.m and
.sup.13C.sup.n in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup.m) and
t.sub.2(.sup.13C.sup.n), and the chemical shift value of
.sup.1H.sup.n in a direct time domain dimension,
t.sub.3(.sup.1H.sup.n), and (2) to sine modulate the chemical shift
evolution of .sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the
chemical shift evolution of .sup.1H.sup.m. Then, the NMR signals
are processed to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift values of .sup.13C.sup.n and .sup.1H.sup.n are
measured in two frequency domain dimensions,
.omega..sub.2(.sup.13C.sup.n) and .omega..sub.3(.sup.1H.sup.n),
respectively, and (2) the chemical shift values of .sup.1H.sup.m
and .sup.13C.sup.m are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.m), by the frequency differences between
each of the two peaks forming each of the anti-phase peak pairs and
the frequencies at the center of the two peaks, respectively, where
the sine-modulated 3D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sub.m in a phase sensitive
manner.
[0017] A further aspect of the present invention relates to a
method of conducting a reduced dimensionality two-dimensional (2D)
HB,CB,(CG,CD),HD nuclear magnetic resonance (NMR) experiment by
measuring the chemical shift values for the following nuclei of a
protein molecule: (1) a .beta.-proton of an amino acid residue with
an aromatic side chain, .sup.1H.sup..beta.; (2) a .beta.-carbon of
an amino acid residue with an aromatic side chain,
.sup.13C.sup..beta.; and (3) a .delta.-proton of an amino acid
residue with an aromatic side chain, .sup.1H.sup..delta., where the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
which are encoded in a peak pair of a 2D NMR spectrum are detected
in a phase sensitive manner. The method involves providing a
protein sample and applying radiofrequency pulses to the protein
sample which effect a nuclear spin polarization transfer where the
chemical shift evolutions of .sup.1H.sup..beta. and
.sup.13C.sup..beta. are connected to the chemical shift evolution
of .sup.1H.sup.67, under conditions effective (1) to generate NMR
signals encoding the chemical shift value of .sup.13C.sup..beta. in
a phase sensitive manner in an indirect time domain dimension,
t.sub.1(.sup.13C.sup..beta.), and the chemical shift value of
.sup.1H.sup..delta. in a direct time domain dimension,
t.sub.2(.sup.1H.sup.67), and (2) to sine modulate the chemical
shift evolution of .sup.13C.sup..beta. in
t.sub.1(.sup.13C.sup..beta.) with the chemical shift evolution of
.sup.1H.sup..beta.. Then, the NMR signals are processed to generate
a sine-modulated 2D NMR spectrum with an anti-phase peak pair
derived from the sine modulating where (1) the chemical shift value
of .sup.1H.sup..delta. is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup..delta.), and (2) the chemical shift
values of .sup.1H.sup..beta. and .sup.13C.sup..beta. are measured
in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming the anti-phase peak pair and the
frequency at the center of the two peaks, respectively, where the
sine-modulated 2D NMR spectrum enables detection of the chemical
shift value of .sup.1H.sup..beta. in a phase sensitive manner.
[0018] The present invention also relates to a method of conducting
a reduced dimensionality two-dimensional (2D) H,C,H-COSY nuclear
magnetic resonance (NMR) experiment by measuring the chemical shift
values for .sup.1H.sup.m, .sup.13C.sup.m, and .sup.1H.sup.n of a
protein molecule where m and n indicate atom numbers of two CH,
CH.sub.2 or CH.sub.3 groups in an amino acid residue, where the
chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m which are
encoded in a peak pair of a 2D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup.m and .sup.13C.sup.m are connected
to the chemical shift evolution of .sup.1H.sup.n, under conditions
effective (1) to generate NMR signals encoding the chemical shift
value of .sup.13C.sup.m in a phase sensitive manner in an indirect
time domain dimension, t.sub.1(.sup.13C.sup.m), and the chemical
shift value of .sup.1H.sup.n in a direct time domain dimension,
t.sub.2(.sup.1H.sup.n), and (2) to sine modulate the chemical shift
evolution of .sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the
chemical shift evolution of .sup.1H.sup.m. Then, the NMR signals
are processed to generate a sine-modulated 2D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift value of .sup.1H.sup.n is measured in a
frequency domain dimension, .omega..sub.2(.sup.1H.sup.n), and (2)
the chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.m), by the frequency differences between
each of the two peaks forming each of the anti-phase peak pairs and
the frequencies at the center of the two peaks, respectively, where
the sine-modulated 2D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sub.m in a phase sensitive
manner.
[0019] Another aspect of the present invention relates to a method
for sequentially assigning chemical shift values of an
.alpha.-proton, .sup.1H.sup..alpha., an .alpha.-carbon,
.sup.13C.sup..alpha., a polypeptide backbone amide nitrogen,
.sup.15N, and a polypeptide backbone amide proton, .sup.1H.sup.N,
of a protein molecule. The method involves providing a protein
sample and conducting a set of reduced dimensionality (RD) nuclear
magnetic resonance (NMR) experiments on the protein sample, where
the chemical shift values of .sup.1H.sup..alpha. and
.sup.13C.sup..alpha. which are encoded in a peak pair of a 3D NMR
spectrum are detected in a phase sensitive manner, including: (1) a
RD three-dimensional (3D) HA,CA,(CO),N,HN NMR experiment to measure
and connect chemical shift values of the .alpha.-proton of amino
acid residue i-1, .sup.1H.sup..alpha..sub.i-1, the .alpha.-carbon
of amino acid residue i-1, .sup.13C.sup..alpha..sub.i-1, the
polypeptide backbone amide nitrogen of amino acid residue i,
.sup.15N.sub.i, and the polypeptide backbone amide proton of amino
acid residue i, .sup.1H.sup.N and (2) a RD 3D HNNCAHA NMR
experiment to measure and connect the chemical shift values of the
.alpha.-proton of amino acid residue i, .sup.1H.sup..alpha..sub.i,
the .alpha.-carbon of amino acid residue i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup..alpha., .sup.13C.sup..alpha.,
.sup.15N, and .sup.1H.sup.N are obtained by (i) matching the
chemical shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 with the chemical shift values of
.sup.1H.sup..alpha..sub.i and .sup.13C.sup..alpha..sub.i, (ii)
using the chemical shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 to identify the type of amino acid
residue i-1, and (iii) mapping sets of sequentially connected
chemical shift values to the amino acid sequence of the polypeptide
chain and using the chemical shift values to locate secondary
structure elements within the polypeptide chain.
[0020] Yet another aspect of the present invention relates to a
method for sequentially assigning chemical shift values of a
.beta.-proton, .sup.1H.sup..beta., a .beta.-carbon,
.sup.13C.sup..beta., an .alpha.-proton, .sup.1H.sup..alpha., an
.alpha.-carbon, .sup.13C.sup..alpha., a polypeptide backbone amide
nitrogen, .sup.15N, and a polypeptide backbone amide proton,
.sup.1H.sup.N, of a protein molecule. The method involves providing
a protein sample and conducting a set of reduced dimensionality
(RD) nuclear magnetic resonance (NMR) experiments on the protein
sample, where the chemical shift values of
.sup.1H.sup..alpha./.beta. and .sup.13C.sup..alpha./.beta. which
are encoded in peak pairs of a 3D NMR spectrum are detected in a
phase sensitive manner, including: (1) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta. (CO)NHN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i-1, .sup.1H.sup..beta..sub.i-1, the
.beta.-carbon of amino acid residue i-1,
.sup.13C.sup..beta..sub.i-1, the .alpha.-proton of amino acid
residue i-1, .sup.1H.sup..alpha..sub.i-1, the .alpha.-carbon of
amino acid residue i-1, .sup.13C.sup..alpha..sub.i-1, the
polypeptide backbone amide nitrogen of amino acid residue i,
.sup.15N.sub.i, and the polypeptide backbone amide proton of amino
acid residue i, .sup.1H.sup.N.sub.i and (2) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta., N,HN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
the .alpha.-proton of amino acid residue i,
.sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid residue
i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup..beta., .sup.13C.sup..beta.,
.sup.1H.sup..alpha., .sup.13C.sup..alpha., .sup.15N, and
.sup.1H.sup.N are obtained by (i) matching the chemical shift
values of the .alpha.- and .beta.-protons of amino acid residue
i-1, .sup.1H.sup..alpha./.beta..sub.i-1, and the .alpha.- and
.beta.-carbons of amino acid residue i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, with the chemical shift values
of .sup.1H.sup..alpha./.beta..sub.i and
.sup.13C.sup..alpha./.beta..sub.i, (ii) using the chemical shift
values of .sup.1H.sup..alpha./.beta..sub.i-1 and
.sup.13C.sup..alpha./.beta..sub.i-1 to identify the type of amino
acid residue i-1, and (iii) mapping sets of sequentially connected
chemical shift values to the amino acid sequence of the polypeptide
chain and using the chemical shift values to locate secondary
structure elements within the polypeptide chain.
[0021] A further aspect of the present invention involves a method
for sequentially assigning chemical shift values of aliphatic
protons, .sup.1H.sup.ali, aliphatic carbons, .sup.13C.sup.ali, a
polypeptide backbone amide nitrogen, .sup.15N, and a polypeptide
backbone amide proton, .sup.1H.sup.N, of a protein molecule. The
method involves providing a protein sample and conducting a set of
reduced dimensionality (RD) nuclear magnetic resonance (NMR)
experiments on the protein sample, where the chemical shift values
of .sup.1H.sup.ali and .sup.13C.sup.ali which are encoded in peak
pairs of a 3D NMR spectrum are detected in a phase sensitive
manner, including: (1) a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment
to measure and connect the chemical shift values of the aliphatic
protons of amino acid residue i-1, .sup.1H.sup.ali.sub.i-1 the
aliphatic carbons of amino acid residue i-1,
.sup.13C.sup.ali.sub.i-1, the polypeptide backbone amide nitrogen
of amino acid residue i, .sup.15N.sub.i, and the polypeptide
backbone amide proton of amino acid residue i, .sup.1H.sup.N.sub.i
and (2) a RD 3D H.sup..alpha./.beta., C.sup..alpha./.beta.,N,HN NMR
experiment to measure and connect the chemical shift values of the
.beta.-proton of amino acid residue i, .sup.1H.sup..beta..sub.i,
the .beta.-carbon of amino acid residue i,
.sup.13C.sup..beta..sub.i, the .alpha.-proton of amino acid residue
i, .sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid
residue i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup.ali, .sup.13C.sup.ali, .sup.15N, and
.sup.1H.sup.N are obtained by (i) matching the chemical shift
values of the .alpha.- and .beta.-protons of amino acid residue
i-1, .sup.1H.sup..alpha./.beta..sub.i-1, and the .alpha.- and
.beta.-carbons of amino acid residue i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, with the chemical shift values
of .sup.1H.sup..alpha./.beta..sub.i and
.sup.13C.sup..alpha./.beta..sub.i of amino acid residue i, (ii)
using the chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 to identify the type of amino acid residue
i-1, and (iii) mapping sets of sequentially connected chemical
shift values to the amino acid sequence of the polypeptide chain
and using the chemical shift values to locate secondary structure
elements within the polypeptide chain.
[0022] The present invention also relates to a method for
sequentially assigning chemical shift values of aliphatic protons,
.sup.1Ha.sup.ali, aliphatic carbons, .sup.13C.sup.ali, a
polypeptide backbone amide nitrogen, .sup.15N, and a polypeptide
backbone amide proton, .sup.1H.sup.N, of a protein molecule. The
method involves providing a protein sample and conducting a set of
reduced dimensionality (RD) nuclear magnetic resonance (NMR)
experiments on the protein sample, where the chemical shift values
of .sup.1H.sup.ali and .sup.13C.sup.ali which are encoded in peak
pairs of a 3D NMR spectrum are detected in a phase sensitive
manner, including: (1) a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment
to measure and connect the chemical shift values of the aliphatic
protons of amino acid residue i-1, .sup.1H.sup.ali.sub.i-1, the
aliphatic carbons of amino acid residue i-1,
.sup.13C.sup.ali.sub.i-1, the polypeptide backbone amide nitrogen
of amino acid residue i, .sup.15N.sub.i, and the polypeptide
backbone amide proton of amino acid residue i, .sup.1H.sup.N and
(2) a RD 3D HNNCAHA NMR experiment to measure and connect the
chemical shift values of the .alpha.-proton of amino acid residue
i, .sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid
residue i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup.ali, .sup.13C.sup.ali, .sup.15N, and
.sup.1H.sup.N are obtained by (i) matching the chemical shift
values of the .alpha.-proton of amino acid residue i-1,
.sup.1H.sup..alpha..sub.i-1, and the .alpha.-carbon of amino acid
residue i-1, .sup.13C.sup..alpha..sub.i-1, with the chemical shift
values of .sup.1H.sup..alpha..sub.i and .sup.13C.sup..alpha..sub.i,
(ii) using the chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 to identify the type of amino acid residue
i-1, and (iii) mapping sets of sequentially connected chemical
shift values to the amino acid sequence of the polypeptide chain
and using the chemical shift values to locate secondary structure
elements within the polypeptide chain.
[0023] Another aspect of the present invention involves a method
for obtaining assignments of chemical shift values of .sup.1H,
.sup.13C and .sup.15N of a protein molecule. The method involves
providing a protein sample and conducting four reduced
dimensionality (RD) nuclear magnetic resonance (NMR) experiments on
the protein sample, where the chemical shift values of .sup.1H and
.sup.13C which are encoded in peak pairs of a 3D NMR spectrum are
detected in a phase sensitive manner and (1) a first experiment is
selected from a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, a
RD 3D HA, CA,(CO),N,HN NMR experiment, or a RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment for obtaining sequential
correlations of chemical shift values; (2) a second experiment is
selected from a RD 3D HNNCAHA NMR experiment, a RD 3D
H.sup..alpha./.beta., C.sup..alpha./.beta.,N,HN NMR experiment, or
a RD 3D HNN<CO,CA> NMR experiment for obtaining intraresidue
correlations of chemical shift values; (3) a third experiment is a
RD 3D H,C,C,H-COSY NMR experiment for obtaining assignments of
sidechain chemical shift values; and (4) a fourth experiment is a
RD 2D HB,CB,(CG,CD),HD NMR experiment for obtaining assignments of
aromatic sidechain chemical shift values.
[0024] The present invention enables one to record an entire suite
of RD NMR experiments used for obtaining chemical shift assignments
and secondary structure determination of a protein molecule in a
manner that enables frequency discrimination for the projected
chemical shift and allows one to place the two peaks forming a peak
pair into separate spectra. The frequency discrimination allows one
to place the radiofrequency carrier in the center of the projected
chemical shift range.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a schematic presentation describing
cosine-modulated and sine-modulated RD NMR data acquisition and
processing, as well as the editing of the peaks forming RD NMR peak
pairs into separate spectra. Data sets (1) and (2) correspond to
real part and imaginary part, respectively, of a cosine-modulated
RD NMR experiment, in which .OMEGA..sub.y is detected in a phase
sensitive manner and the evolution of the chemical shift
.OMEGA..sub.x effects a cosine modulation. Data sets (3) and (4)
correspond to real part and imaginary part, respectively, of a
sine-modulated RD NMR experiment, in which .OMEGA..sub.y is
detected in a phase sensitive manner and the evolution of
.OMEGA..sub.x effects a sine modulation, further enabling the phase
sensitive detection of .OMEGA..sub.x. The two data sets as well as
the corresponding cosine and sine functions for the
cosine-modulated RD NMR and sine-modulated RD NMR are shown in (A)
and (B), respectively. Forming the sum and the difference of the
cosine-modulated and sine-modulated RD NMR spectra allows one to
edit the two peaks forming the peak pairs into separate spectra.
The data sets that need to be linearly combined in the time domain
to achieve such editing are shown in (C) and (D).
[0026] FIGS. 2A-K show the polarization transfer pathways (top) and
stick diagrams of the in-phase peak pattern observed along
.omega..sub.1(.sup.13C) (bottom) for the cosine-modulated RD NMR
experiments implemented for the present invention (the 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN experiment, the 3D
HACA(CO)NHN experiment, the 3D HC(C-TOCSY-CO)NHN experiment, the 3D
HNNCAHA experiment, the 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.COHA experiment, the 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.NHN experiment, the 3D
HNN<CO,CA> experiment, the 3D HCCH-COSY experiment, the 3D
HCCH-TOCSY experiment, the 2D HBCB(CGCD)HD experiment, and the 2D
.sup.1H-TOCSY-relayed-HCH-COSY experiment, respectively). For the
sine-modulated RD NMR experiments, anti-phase peak pairs are
observed (see FIG. 1) instead of the in-phase peak pattern for the
cosine-modulated RD NMR experiments. The boxes comprise nuclei
whose chemical shifts are measured in the common dimension
.omega..sub.1, and the nuclei which are detected in quadrature in
t.sub.1 are marked with an asterisk. Bold solid and hatched boxes
indicate intraresidue and sequential connectivities, respectively,
and the resulting signals sketched in the stick diagrams are
represented accordingly. Those .sup.13C nuclei whose magnetization
is used to detect central peaks (Szyperski et al., J. Am. Chem.
Soc., 118:8146-8147 (1996), which is hereby incorporated by
reference in its entirety), as well as the resulting subspectrum II
shown at the bottom are highlighted in grey. The magnetization is
frequency labeled with single-quantum coherence of the encircled
nuclei during t.sub.2 and detected on the boxed protons. Except for
FIG. 2G, the in-phase splittings 2.DELTA..OMEGA.(.sup.1H) are equal
to
2.kappa..delta..OMEGA.(.sup.1H)[.gamma.(.sup.1H)/.gamma.(.sup.13C)],
where .kappa., .delta..OMEGA.(.sup.1H) and .gamma.(X) denote the
scaling factor applied for .sup.1H chemical shift evolution (set to
1.0 for the present study), the chemical shift difference with
respect to the apparent .sup.1H carrier position, and the
gyromagnetic ratio of nucleus X, respectively. In FIG. 2G, the
in-phase splittings 2.DELTA..OMEGA.(.sup.13C.sup..alpha.) are equal
to 2.kappa..delta..OMEGA.(.sup.13C.sup..alpha.), where .kappa. and
.delta..OMEGA.(.sup.13C.sup..alpha.) are the scaling factor applied
for .sup.13C.sup..alpha. chemical shift evolution.sup.13 (set to
0.5 for the present study) and the chemical shift difference with
respect to the apparent .sup.13C.sup..alpha. carrier position,
respectively.
[0027] FIG. 3A illustrates the experimental scheme for the 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN experiment.
Rectangular 90.degree. and 180.degree. pulses are indicated by thin
and thick vertical bars, respectively, and phases are indicated
above the pulses. Where no radio-frequency (r.f.) phase is marked,
the pulse is applied along x. In order to accomplish sine
modulation, .phi..sub.1=y. The scaling factor .kappa. for .sup.1H
chemical shift evolution during t.sub.1 is set to 1.0. The
high-power 90.degree. pulse lengths were: 5.9 .mu.s for .sup.1H,
15.4 .mu.s for .sup.13C, and 38 .mu.s for .sup.15N. Pulses on
.sup.13C prior to t.sub.1(.sup.13C) are applied at high power, and
.sup.13C decoupling during t.sub.1(.sup.1H) is achieved using a
(90.sub.x-180.sub.y-90.sub.x) composite pulse. Subsequently, the
90.degree. and 180.degree. pulse lengths applied for
.sup.13C.sup..alpha./.beta. are adjusted to 47.5 .mu.s and 42.5
.mu.s, respectively, to minimize perturbation of .sup.13CO spins.
The width of the 90.degree. pulse applied on .sup.13CO pulse is 52
.mu.s and the corresponding 180.degree. pulses are applied with
same power. A SEDUCE 180.degree. pulse with a length of 200 .mu.s
is used to decouple .sup.13CO during t.sub.1 and .tau..sub.4. The
length of the spin-lock purge pulses SL.sub.x and SL.sub.y are 1.2
ms and 0.6 ms, respectively. WALTZ16 is employed to decouple
.sup.1H (r.f. field strength=9.2 kHz) during the heteronuclear
magnetization transfers as well as to decouple .sup.15N during
acquisition (r.f.=1.78 kHz). The SEDUCE sequence is used for
decoupling of .sup.13C.sup..alpha. during .sup.15N evolution period
(rf=1.0 kHz). The .sup.1H r.f. carrier is placed at 0 ppm before
the start of the semi constant time .sup.1H chemical shift
evolution period, and then switched to the water line at 4.78 ppm
after the second 90.degree. .sup.1H pulse. Initially, the .sup.13C
and .sup.15N r. f. carriers are set to 43 ppm and 120.9 ppm,
respectively. The .sup.13C carrier is set to 56 ppm during the
second .tau..sub.4/2 delay. The duration and strengths of the
pulsed z-field gradients (PFGs) are: G1 (1 ms, 24 G/cm); G2 (100
.mu.s, 16 G/cm); G3 (250 .mu.s, 29.5 G/cm); G4 (250 .mu.s, 30
G/cm); G5 (1.5 ms, 20 G/cm); G6 (1.25 ms, 30 G/cm); G7 (500 .mu.s,
8 G/cm); G8 (125 .mu.S, 29.5 G/cm). All PFG pulses are of
rectangular shape. A recovery delay of at least 100 .mu.s duration
is inserted between a PFG pulse and an r.f. pulse. The delays are:
.tau..sub.1=800 .mu.s, .tau..sub.2=3.1 ms, .tau..sub.3=3.6 ms,
.tau..sub.4=7.2 ms, .tau..sub.5=4.4 ms, .tau..sub.6=24.8 ms,
.tau..sub.7=24.8 ms, .tau..sub.8=5.5 ms, .tau..sub.9=4.6 ms,
.tau..sub.10=1.0 ms. .sup.1H-frequency labeling is achieved in a
semi constant-time fashion with t.sub.1.sup.a(0)=1.7 ms,
t.sub.1.sup.b(0)=1 .mu.s, t.sub.1.sup.c(0)=1.701 ms,
.DELTA.t.sub.1.sup.a=33.3 .mu.s, .DELTA.t.sub.1.sup.b=19.3 .mu.s,
.DELTA.t.sub.1.sup.c=-14 .mu.s. Hence, the fractional increase of
the semi constant-time period with t.sub.1.sup.a equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a=0.58. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x, x, -x, -x; .phi..sub.3=x,
-x; .phi..sub.4=x, -x; .phi..sub.5=x; .phi..sub.6 x, x, -x, -x;
.phi..sub.7=x; .phi..sub.8 (receiver)=x, -x, -x, x. The sensitivity
enhancement scheme of Kay (Cavanagh et al., Protein NMR
Spectroscopy, Academic Press, San Diego, (1996), which is hereby
incorporated by reference in its entirety) is employed, i.e., the
sign of G.sub.6 is inverted in concert with a 180.degree. shift of
.phi..sub.7. Quadrature detection in t.sub.1(.sup.13C) and
t.sub.2(.sup.15N) is accomplished by altering the phases
.phi..sub.2 and .phi..sub.5, respectively, according to States-TPPI
(Cavanagh et al., Protein NMR Spectroscopy, Academic Press, San
Diego, (1996), which is hereby incorporated by reference in its
entirety). For acquisition of central peaks derived from .sup.13C
steady state magnetization, a second data set with .phi..sub.1=-x
is collected. The sum and the difference of the two resulting data
sets generate subspectra II and I, respectively, containing the
central peaks and peak pairs.
[0028] FIG. 3B illustrates the experimental scheme for the 3D
HACA(CO)NHN experiment. Rectangular 90.degree. and 180.degree.
pulses are indicated by thin and thick vertical bars, respectively,
and phases are indicated above the pulses. Where no radio-frequency
(r.f.) phase is marked, the pulse is applied along x. In order to
accomplish sine modulation, .phi..sub.1=y. The scaling factor
.kappa. for .sup.1H chemical shift evolution during t.sub.1 is set
to 1.0. The high power 90.degree. pulse lengths were: 5.8 .mu.s for
.sup.1H and 15.4 .mu.s for .sup.13C, and 38 .mu.s for .sup.15N.
Pulses on .sup.13C prior to t.sub.1(.sup.13C) are applied at high
power, and .sup.13C decoupling during t.sub.1(.sup.1H) is achieved
using a (90.sub.x-180.sub.y-90.sub.x) composite pulse.
Subsequently, the 90.degree. and 180.degree. pulse lengths of
.sup.13C.sup..alpha. are adjusted to 51.5 .mu.s and 46 .mu.s,
respectively, to minimize perturbation of the .sup.13CO spins. The
width of the 90.degree. pulses applied to .sup.13CO pulse is 52
.mu.s and the corresponding 180.degree. pulses are applied with
same power. A SEDUCE 180.degree. pulse with a length 252 .mu.s is
used to decouple .sup.13CO during t.sub.1. The length of the
spin-lock purge pulses SL.sub.x and SL.sub.y are 2.5 ms and 1 ms,
respectively. WALTZ16 is employed to decouple .sup.1H (r.f. field
strength=9.2 kHz) during the heteronuclear magnetization transfers
as well as to decouple .sup.15N during acquisition (r.f.=1.78 kHz).
The SEDUCE sequence is used for decoupling of .sup.13C.sup..alpha.
during the .sup.15N chemical shift evolution period (r.f.=1.0 kHz).
The .sup.1H r.f. carrier is placed at 0 ppm before the start of the
semi constant time .sup.1H evolution period, and then switched to
the water line at 4.78 ppm after the second 90.degree. .sup.1H
pulse. The .sup.13C.sup..alpha. and .sup.15N r.f. carriers are set
to 56.1 ppm and 120.9 ppm, respectively. The duration and strengths
of the pulsed z-field gradients (PFGs) are: G1 (1 ms, 24 G/cm); G2
(100 .mu.s, 16 G/cm); G3 (1 ms, 24 G/cm); G4 (250 .mu.s, 30 G/cm);
G5 (1.5 ms, 20 G/cm); G6 (1.25 ms, 30 G/cm); G7 (500 .mu.s, 8
G/cm); G8 (125 .mu.s, 29.5 G/cm). All PFG pulses are of rectangular
shape. A recovery delay of at least 100 .mu.s duration is inserted
between a PFG pulse and an r.f. pulse. The delays are:
.tau..sub.1=1.6 ms, .tau..sub.2=3.6 ms, .tau..sub.3=4.4 ms,
.tau..sub.4=.tau..sub.5=24.8 ms, .tau..sub.6=5.5 ms,
.tau..sub.7=4.6 ms, .tau..sub.8=1 ms. .sup.1H-frequency labeling is
achieved in a semi constant-time fashion with t.sub.1.sup.a(0)=1.7
ms, t.sub.1.sup.b(0)=1 .mu.s, t.sub.1.sup.c(0)=1.701 ms,
.DELTA.t.sub.1.sup.a=60 .mu.s, .DELTA.t.sub.1.sup.b=35.4 .mu.s,
.DELTA.t.sub.1.sup.c=24.6 .mu.s. Hence, the fractional increase of
the semi constant-time period with t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a=0.58. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x, x, -x, -x; .phi..sub.3=x,
-x; .phi..sub.4=x; .phi..sub.5=x, x, -x, -x; .phi..sub.6=x;
.phi..sub.7(receiver)=x, -x, -x, x. The sensitivity enhancement
scheme of Kay (Cavanagh et al., Protein NMR Spetroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) is employed., i.e., the sign of G6 is inverted in
concert with a 180.degree. shift of .phi..sub.6. Quadrature
detection in t.sub.1(.sup.13C) and t.sub.2(.sup.15N) is
accomplished by altering the phases .phi..sub.2 and .phi..sub.4,
respectively, according to States-TPPI (Cavanagh et al., Protein
NMR Spectroscopy, Academic Press, San Diego, (1996), which is
hereby incorporated by reference in its entirety). For acquisition
of central peaks derived from .sup.13C steady state magnetization,
a second data set with .phi..sub.1=-x is collected. The sum and the
difference of the two resulting data sets generate subspectra II
and I, respectively, containing the central peaks and peak
pairs.
[0029] FIG. 3C illustrates the experimental scheme for the 3D
HC(C-TOCSY-CO)NHN experiment. Rectangular 90.degree. and
180.degree. pulses are indicated by thin and thick vertical bars,
respectively, and phases are indicated above the pulses. Where no
radio-frequency (r.f.) phase is marked, the pulse is applied along
x. In order to accomplish sine modulation, .phi..sub.1=y. The
scaling factor .kappa. for .sup.1H chemical shift evolution during
t.sub.1 is set to 1.0. The high power 90.degree. pulse lengths
were: 5.8 .mu.s for .sup.1H and 15.5 .mu.s for .sup.13C, and 38
.mu.s for .sup.15N. Pulses on .sup.13C prior to t.sub.1(.sup.13C)
are applied at high power, and .sup.13C decoupling during
t.sub.1(.sup.1H) is achieved using a (90.sub.x-180.sub.y-90.sub.x)
composite pulse. Subsequently, the 90.degree. and 180.degree. pulse
lengths applied for .sup.13C are adjusted to 47.0 .mu.s and 42.5
.mu.s, respectively, to minimize perturbation of .sup.13CO spins.
The width of the 90.degree. pulses applied to .sup.13CO pulse is 52
.mu.s and the corresponding 180.degree. pulses are applied with
same power. A SEDUCE 180.degree. pulse with a length 200 .mu.s is
used to decouple .sup.13CO during t.sub.1 and .tau..sub.4 period.
WALTZ16 is employed to decouple .sup.1H (r.f. field strength=9.2
kHz) during the heteronuclear magnetization transfers as well as to
decouple .sup.15N during acquisition (r.f.=1.78 kHz). The SEDUCE
sequence is used for decoupling of .sup.13C.sup..alpha. during the
.sup.15N chemical shift evolution period (r.f.=1.0 kHz). The
.sup.1H r.f. carrier is placed at 0 ppm before the start of the
semi constant time .sup.1H evolution period, and then switched to
the water line at 4.78 ppm after the second 90.degree. .sup.1H
pulse. The .sup.13C and .sup.15N r.f. carriers are set to 43 ppm
and 120.9 ppm, respectively. The lengths of the .sup.13C spin-lock
purge pulses, SL.sub.x, are 2.5 ms and 1.25 ms, respectively,
before and after the carbon-carbon total correlation spectroscopy
(TOCSY) relay. .sup.13C isotropic mixing is accomplished using
DIPSI-2 scheme with a r.f. field strength of 8.5 kHz. The duration
and strengths of the pulsed z-field gradients (PFGs) are: G1 (2 ms,
30 G/cm); G2 (100 .mu.s, 8 G/cm); G3 (200 .mu.s, 4 G/cm); G4 (2 ms,
30 G/cm); G5(1.25 ms, 30 G/cm); G6 (500 .mu.s, 5 G/cm); G7 (125
.mu.s, 29.5 G/cm). All PFG pulses are of rectangular shape. A
recovery delay of at least 100 .mu.s duration is inserted between a
PFG pulse and an r.f. pulse. The delays are: .tau..sub.1=950 .mu.s,
.tau..sub.2=3.1 ms, .tau..sub.3=3.6 ms, .tau..sub.4=7.2 ms,
.tau..sub.5=4.45 ms, .tau..sub.6=24.8 ms, .tau..sub.7=24.8 ms,
.tau..sub.8=5.5 ms, .tau..sub.9=4.8 ms, .tau..sub.10=1 ms.
.sup.1H-frequency labeling is achieved in a semi constant-time
fashion with t.sub.1.sup.a(0)=1.7 ms, t.sub.1.sup.b(0)=1 .mu.S,
t.sub.1.sup.c(0)=1.701 ms, .DELTA.t.sub.1.sup.a=33.3 .mu.s,
.DELTA.t.sub.1.sup.a=19.3 .mu.s, .DELTA.t.sub.1.sup.c=-14 .mu.s.
Hence, the fractional increase of the semi constant-time period
with t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a=0.58. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x, =x; .phi..sub.3=x, x, -x,
-x; .phi..sub.4=x, -x; .phi..sub.5=x, x, -x, -x; .phi..sub.6=x, x,
-x, -x; .phi..sub.7=x; .phi..sub.8=4x,4(-x); .phi..sub.9=x;
.phi..sub.10(receiver) x, -x, -x, x. The sensitivity enhancement
scheme of Kay (Cavanagh et al., Protein NMR Spectroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) is employed, i.e., the sign of G5 is inverted in
concert with a 180.degree. shift of .phi..sub.9. Quadrature
detection in t.sub.1(.sup.13C) and t.sub.2(.sup.15N) is
accomplished by altering the phases .phi..sub.2 and .phi..sub.7,
respectively, according to States-TPPI (Cavanagh et al., Protein
NMR Spectroscopy, Academic Press, San Diego, (1996), which is
hereby incorporated by reference in its entirety). For acquisition
of central peaks derived from .sup.13C steady state magnetization,
a second data set with .phi..sub.1=-x is collected. The sum and the
difference of the two resulting data sets generate subspectra II
and I, respectively, containing the central peaks and peak
pairs.
[0030] FIG. 3D illustrates the experimental scheme for the 3D
HNNCAHA experiment. Rectangular 90.degree. and 180.degree. pulses
are indicated by thin and thick vertical bars, respectively, and
phases are indicated above the pulses. Where no radio-frequency
(r.f.) phase is marked, the pulse is applied along x. In order to
accomplish sine modulation, .phi..sub.8=y. The scaling factor
.kappa. for .sup.1H chemical shift evolution during t.sub.1 is set
to 1.0. The 90.degree. pulse lengths were: 5.8 .mu.s for .sup.1H
and 21.6 .mu.s for .sup.13C.sup..alpha., and 38 .mu.s for .sup.15N,
where the 90.degree. pulse width for .sup.13C.sup..alpha. is
adjusted to generate a null of excitation in the center of the CO
chemical shift range. The selective 90.degree. .sup.1H pulse used
to flip back the water magnetization is applied for the 1.8 ms with
the SEDUCE-1 profile. WALTZ16 is employed to decouple .sup.1H (r.f.
field strength=9.2 kHz) during the heteronuclear magnetization
transfers as well as to decouple of .sup.15N (r.f.=1.78 kHz) during
acquisition. SEDUCE is used for decoupling of .sup.13CO (max.
r.f.=3.0 kHz). WURST-2 is used for simultaneous band selective
decoupling of .sup.13CO and .sup.13C.sup..beta. during .tau..sub.4
and the .sup.1H and .sup.13C chemical shift evolution during
t.sub.1. 3.0 kHz sweeps at 176 ppm and 30 ppm, respectively, are
used for decoupling of .sup.13C.sup..beta.and .sup.13C.sup..beta.
(except for Ser, Thr, Ala). A sweep of 600 Hz is used at 14 ppm to
decouple .sup.13C.sup..beta. of Ala. The .sup.1H r.f. carrier is
placed at the position of the solvent line at 4.78 ppm for the
first three .sup.1H pulses and the first WALTZ period, then
switched to 0 ppm during the first delay .tau..sub.4/2, and
subsequently switched back to the water line at 4.78 ppm during
t.sub.1.sup.c. The .sup.13C.sup..alpha. and .sup.15N carriers are
set to 56.1 ppm and 120.9 ppm, respectively. The duration and
strengths of the pulsed z-field gradients (PFGs) are: G1 (500
.mu.s, 8 G/cm); G2 (500 .mu.s, 4 G/cm); G3 (1 ms, 30 G/cm); G4 (150
.mu.s, 25 G/cm); G5 (1.25 ms, 30 G/cm); G6 (500 .mu.s, 8 G/cm); G7
(125 .mu.s, 29.57 G/cm). All PFG pulses are of rectangular shape. A
recovery delay of at least 100 .mu.s duration is inserted between a
PFG pulse and an r.f. pulse. The delays have the following values:
.tau..sub.1=4.6 ms, .tau..sub.2=5.5 ms, .tau..sub.3=24 ms,
.tau..sub.4=2.0 ms, .tau..sub.5=500 .mu.s. .sup.13C-frequency
labeling is achieved in a semi constant-time fashion with
t.sub.1.sup.a(0)=1.065 ms, t.sub.1.sup.b(0)=49 .mu.s,
t.sub.1.sup.c(0)=984 .mu.s, .DELTA.t.sub.1.sup.a=65 .mu.s,
.DELTA.t.sub.1.sup.b=49 .mu.s, .DELTA.t.sub.1.sup.c=-16 .mu.s.
Hence, the fractional increase of the semi constant-time period
with t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a=0.76. Note that
the acquisition starts with the second complex point in t.sub.1,
while the first one is obtained by linear prediction. This ensures
that a zero first-order phase correction is achieved along
.omega..sub.1. Phase cycling: .phi..sub.1=x, -x; .phi..sub.2=x, x,
-x, -x; .phi..sub.3=x, -x, -x, x; .phi..sub.4=x; .phi..sub.5=4(x),
4(-x); .phi..sub.6=x; .phi..sub.7(receiver)=x, -x, -x, x. The
sensitivity enhancement scheme of Kay (Cavanagh et al., Protein NMR
Spetroscopy, Academic Press, San Diego, (1996), which is hereby
incorporated by reference in its entirety) is employed, i.e., the
sign of G5 is inverted in concert with a 180.degree. shift of
.phi..sub.6. Quadrature detection in t.sub.1(.sup.13C) and
t.sub.2(.sup.15N) is accomplished by altering the phases
.phi..sub.2 and .phi..sub.4 according to States-TPPI (Cavanagh et
al., Protein NMR Spectroscopy, Academic Press, San Diego, (1996),
which is hereby incorporated by reference in its entirety).
[0031] FIG. 3E illustrates the experimental scheme for the 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.COHA experiment.
Rectangular 90.degree. and 180.degree. pulses are indicated by thin
and thick vertical bars, respectively, and phases are indicated
above the pulses. Where no radio-frequency (r.f.) phase is marked,
the pulse is applied along x. In order to accomplish sine
modulation, .phi..sub.1=y. The scaling factor .kappa. for the
.sup.1H chemical shift evolution during t.sub.1 is set to 1.0. The
high power 90.degree. pulse lengths were: 5.9 .mu.s for .sup.1H,
15.4 .mu.s for .sup.13C, and 38.2 .mu.s for .sup.15N. The
90.degree. and 180.degree. pulse lengths of
.sup.13C.sup..alpha./.beta. were adjusted to 47.4 .mu.s and 42.4
.mu.s, respectively, to minimize perturbation of .sup.13CO spins. A
200 .mu.s 180.degree. pulse with SEDUCE profile is used to
selectively invert .sup.13CO magnetization prior to the start of
t.sub.1. The 90.degree. and 180.degree. pulses employed for
excitation of .sup.13CO and subsequent magnetization transfer back
to .sup.13C.sup..alpha. are of rectangular shape and 52 .mu.s and
103 .mu.s duration, respectively. The length of the spin-lock purge
pulses SL.sub.x and SL.sub.y are 2.5 ms and 1 ms, respectively.
WALTZ16 is employed to decouple .sup.1H (r.f. field strength=9.2
kHz) during the heteronuclear magnetization transfers, and for
decoupling of .sup.15N (r.f.=1.78 kHz) during acquisition. GARP is
used for decoupling of .sup.13C.sup..alpha. (r.f.=2.5 kHz). The
.sup.1H r.f. carrier is placed at the position of the solvent line
at 0 ppm before the start of the first semi constant time .sup.1H
evolution period and then switched to the water line at 4.78 ppm
after the second 90.degree. .sup.1H pulse. Initially, the .sup.13C
and .sup.15N r.f. carriers are set to 43 ppm and 120.9 ppm
respectively. The duration and strengths of the pulsed z-field
gradients (PFGs) are: G1=G2 (100 .mu.s, 15 G/cm); G3 (2 ms, 25
G/cm); G4 (100 .mu.s, 10 G/cm); G5 (1 ms, 27 G/cm); G6 (3 ms, 30
G/cm); G7 (1.3 ms, 20 G/cm); G8 (130 .mu.s, 14 G/cm). All PFG
pulses are of rectangular shape. A recovery delay of at least 100
.mu.s duration is inserted between a PFG pulse and an r.f. pulse.
The delays are: .tau..sub.1=800 .mu.s, .tau..sub.2=2.8 ms,
.tau..sub.3=3.6 ms, .tau..sub.4=6.5 ms, .tau..sub.5=1.8 ms,
.tau..sub.6=1 ms, .tau..sub.7=2.8 ms, .tau..sub.8=3.6 ms.
.sup.1H-frequency labeling is achieved in a semi constant-time
fashion with t.sub.1.sup.a(0)=1.7 ms, t.sub.1.sup.b(0)=1 .mu.s,
t.sub.1.sup.c(0)=1.701 ms, .DELTA.t.sub.1.sup.a=33.3 .mu.s,
.DELTA.t.sub.1.sup.b=19.3 .mu.s, .DELTA.t.sub.1.sup.c=-14 .XI.s.
Hence, the fractional increase of the semi constant-time period
with t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a=0.58. Phase
cycling: .phi..sub.1=x; .phi..sub.x=x, -x; .phi..sub.3=x, -x, x,
-x; .phi..sub.4 x; .phi..sub.5(receiver)=x, -x. Quadrature
detection in t.sub.1(.sup.13C) and t.sub.2(.sup.15N) is
accomplished by altering the phases .phi..sub.2 and .phi..sub.4,
respectively, according to States-TPPI (Cavanagh et al., Protein
NMR Spectroscopy, Academic Press, San Diego, (1996), which is
hereby incorporated by reference in its entirety). For acquisition
of central peaks derived from .sup.13C steady state magnetization,
a second data set with .phi..sub.1=-x is collected. The sum and the
difference of the two resulting data sets generate subspectra II
and I, respectively, containing the central peaks and peak pairs
(Szyperski et al., J. Am. Chem. Soc., 118:8146-8147 (1996), which
is hereby incorporated by reference in its entirety).
[0032] FIG. 3F illustrates the experimental scheme for the 3D
H.sup..alpha./.beta.C.sup..alpha./.beta. NHN experiment.
Rectangular 90.degree. and 180.degree. pulses are indicated by thin
and thick vertical bars, respectively, and phases are indicated
above the pulses. Where no radio-frequency (r.f.) phase is marked,
the pulse is applied along x. In order to accomplish sine
modulation, .phi..sub.1=y. The scaling factor .kappa. for the
.sup.1H chemical shift evolution during t.sub.1 is set to 10. The
high power 90.degree. pulse lengths were: 5.9 .mu.s for .sup.1H and
15.4 .mu.s for .sup.13C, and 38 .mu.s for .sup.15N. Pulses on
.sup.13C prior to t.sub.1(.sup.13C) are applied at high power, and
.sup.13C decoupling during t.sub.1(.sup.1H) is achieved using a
(90.sub.x-180.sub.y-90.sub.x) composite pulse. Subsequently, the
90.degree. and 180.degree. pulse lengths of
.sup.13C.sup..alpha./.beta. are adjusted to 49 .mu.s and 43.8 .mu.s
to minimize perturbation of .sup.13CO spins. SEDUCE 180.degree.
pulses of 200 .mu.s pulse length are used to decouple
.sup.13C.sup..beta.. WALTZ16 is employed to decouple .sup.1H (r.f.
field strength=9.2 kHz) during the heteronuclear magnetization
transfers, as well as to decouple .sup.15N (r.f.=1.78 kHz). The
.sup.1H carrier is placed at the position of the solvent line at 0
ppm during the first semi constant time .sup.1H evolution period,
and then switched to the water line 4.78 ppm after the second
90.degree. .sup.1H pulse. The .sup.13C and .sup.15N r.f. carriers
are set to 43 ppm and 120.9 ppm, respectively. The duration and
strengths of the pulsed z-field gradients (PFGs) are: G1 (1 ms, 24
G/cm); G2 (500 .mu.s, 8 G/cm); G3 (250 .mu.s, 15 G/cm); G4 (1 ms,
11 G/cm); G5 (500 .mu.s, 20 G/cm); G6(500 .mu.s, 4 G/cm); G7 (125
.mu.s, 29.5 G/cm). All PFG pulses are of rectangular shape. The
delays are: .tau..sub.1=800 .mu.s, .tau..sub.2=2.8 ms,
.tau..sub.3=3.3 ms, .tau..sub.4=7.2 ms, .tau..sub.5=24 ms,
.tau..sub.6=5.4 ms, .tau..sub.7=4.8 ms, .tau..sub.8=1 ms.
.sup.1H-frequency labeling is achieved in a semi constant-time
fashion with t.sub.1.sup.a(0)=1.7 ms, t.sub.1.sup.b(0)=1 .mu.s,
t.sub.1.sup.c(0)=1.701 ms, .DELTA.t.sub.1.sup.a=33.3 .mu.s,
.DELTA.t.sub.1.sup.b=19.3 .mu.s, .DELTA.t.sub.1=-14 .mu.s. Hence,
the fractional increase of the semi constant-time period with
t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a=0.58. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x; .phi..sub.3=x, -x;
.phi..sub.4=x, x, -x, -x; .phi..sub.5=x; .phi..sub.6(receiver)=x,
-x. The sensitivity enhancement scheme of Kay (Cavanagh et al.,
Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which
is hereby incorporated by reference in its entirety) is employed,
i.e., the sign of G5 is inverted in concert with a 180.degree.
shift of .phi..sub.5. Quadrature detection in t.sub.1(.sup.13C) and
t.sub.2(.sup.15N) is accomplished by altering the phases
.phi..sub.2 and .phi..sub.3, respectively, according to
States-TPPI. For acquisition of central peaks derived from .sup.13C
steady state magnetization, a second data set with .phi..sub.1=-x
is collected. The sum and the difference of the two resulting data
sets generate subspectra II and I, respectively, containing the
central peaks and peak pairs.
[0033] FIG. 3G illustrates the experimental scheme for the 3D
HNN<CO,CA> experiment. Rectangular 900 and 180.degree. pulses
are indicated by thin and thick vertical bars, respectively, and
phases are indicated above the pulses. Where no radio-frequency
(r.f.) phase is marked, the pulse is applied along x. In order to
accomplish sine modulation, .phi..sub.3=y. The scaling factor
.kappa. for .sup.13C.sup..alpha. chemical shift evolution during
t.sub.2 is set to 0.5. The high power 90.degree. pulse lengths
were: 5.8 .mu.s for .sup.1H and 38.5 .mu.s for .sup.15N. The
90.degree. and 180.degree. pulse lengths of .sup.13C.sup..alpha.
were adjusted 54 .mu.s and 48.8 .mu.s to minimize perturbation of
.sup.13CO spins. The length of the 90.degree. pulses applied on
.sup.13CO are 102 .mu.s, and they possess the shape of a sinc
center lobe. The corresponding 180.degree. pulses are applied with
same power and shape. The selective .sup.1H 90.degree. pulse used
for flip-back of water magnetization is applied for 1.8 ms with the
SEDUCE-1 profile. WALTZ16 is employed to decouple .sup.1H (r.f.
field strength=9.2 kHz) during the heteronuclear magnetization
transfers as well as to decouple .sup.15N during acquisition
(r.f.=1.78 kHz). The SEDUCE sequence is used for decoupling of
.sup.13C.sup..alpha. during .sup.15N evolution period (r.f.=0.9
kHz). The .sup.13C.sup..alpha. and .sup.15N r.f. carriers are set
to 176.5 ppm and 120.9 ppm respectively. The duration and strengths
of the pulsed z-field gradients (PFGs) are: G1 (500 .mu.s, 30
G/cm); G2 (500 .mu.s, 5 G/cm); G3 (2 ms, 13 G/cm); G4 (750 .mu.s,
20 G/cm); G5 (200 .mu.s, 5 G/cm); G6 (100 .mu.s, 12 G/cm); G7 (1.25
ms, 30 G/cm); G8 (300 .mu.s, 5 G/cm); G9 (200 .mu.s, 10 G/cm); G10
(125 .mu.s, 29.5 G/cm). All PFG pulses are of rectangular shape. A
recovery delay of at least 100 .mu.s duration is inserted between a
PFG pulse and an r.f. pulse. The delays are: .tau..sub.1=4.6 ms,
.tau..sub.2=5.5 ms, .tau..sub.3=14=28 ms, .tau..sub.5=1 ms. Phase
cycling: .phi..sub.1=x x, -x, -x; .phi..sub.2=x, -x; .phi..sub.3=x;
.phi..sub.4=x; .phi..sub.5=4(x), 4(-x); .phi..sub.6=x;
.phi..sub.7(receiver)=x, -x, -x, x. The sensitivity enhancement
scheme of Kay (Cavanagh et al., Protein NMR Spectroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) is employed, i.e., the sign of G5 is inverted in
concert with a 180.degree. shift of .phi..sub.6. Quadrature
detection in t.sub.1(.sup.13C) and t.sub.2(.sup.15N) is
accomplished by altering the phases .phi..sub.2 and .phi..sub.4,
respectively, according to States-TPPI (Cavanagh et al., Protein
NMR Spectroscopy, Academic Press, San Diego, (1996), which is
hereby incorporated by reference in its entirety). To shift the
apparent .sup.13C.sup..alpha. carrier position to 82.65 ppm i.e.,
downfield to all .sup.13C.sup..alpha. resonances, .phi..sub.3 is
incremented in 60.degree. steps according to TPPI. Note, that the
acquisition was started with the ninth complex point and the first
eight complex points along .omega..sub.1(.sup.13C.sup..beta.) were
obtained by linear prediction. This ensures that a zero first-order
phase correction is achieved along .omega..sub.1 (Szyperski et al.,
J. Magn. Reson., B 108: 197-203 (1995), which is hereby
incorporated by reference in its entirety).
[0034] FIG. 3H illustrates the experimental scheme for the 3D
HCCH-COSY experiment. Rectangular 90.degree. and 180.degree. pulses
are indicated by thin and thick vertical bars, respectively, and
phases are indicated above the pulses. Where no radio-frequency
(r.f.) phase is marked, the pulse is applied along x. In order to
accomplish sine modulation, .phi..sub.1=y. The scaling factor
.kappa. for .sup.1H chemical shift evolution during t.sub.1 is set
to 1.0. The high power 90.degree. pulse lengths were: 5.8 .mu.s for
.sup.1H and 15.4 .mu.s for .sup.13C, and 38 .mu.s for .sup.15N. The
lengths of the .sup.1H spin-lock purge pulses are: first SL.sub.x,
2.8 ms; second SL.sub.x, 1.7 ms; SL.sub.y: 4.9 ms. SEDUCE is used
for decoupling of .sup.13CO during t.sub.1 and t.sub.2 (r.f. field
strength=1 kHz). WURST is used for decoupling of .sup.13C during
acquisition. The .sup.1H carrier is placed at the position of the
solvent line at 0 ppm before the start of the first semi constant
time .sup.1H evolution period, and then switched to the water line
at 4.78 ppm after the second 90.degree. .sup.1H pulse. The .sup.13C
and .sup.15N r.f. carriers are set to 38 ppm and 120.9 ppm
respectively. The duration and strengths of the pulsed z-field
gradients (PFGs) are: G1 (500 .mu.s, 6 G/cm); G2 (500 .mu.s, 7
G/cm); G3 (100 .mu.s, 12 G/cm); G4 (100 .mu.s, 12.5 G/cm); G5 (2
ms, 9 G/cm); G6 (500 .mu.s, 5 G/cm); G7 (1.5 ms, 8 G/cm); G8 (400
.mu.s, 6 G/cm). All gradients are applied along z-axis and are of
rectangular shape. All PFG pulses are of rectangular shape. A
recovery delay of at least 100 .mu.s duration is inserted between a
PFG pulse and an r.f. pulse. The delays are: .tau..sub.1=1.6 ms,
.tau..sub.2=850 .mu.s, .tau..sub.3=2.65 ms, .tau..sub.4=3.5 ms,
.tau..sub.5=7 ms, .tau..sub.6=1.6 ms, .tau..sub.7=3.2 ms. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x, -x; .phi..sub.3=x, -x;
.phi..sub.4=x; .phi..sub.5(receiver)=x, -x. Quadrature detection in
t.sub.1(.sup.13C) and t.sub.2(.sup.13C) is accomplished by altering
the phases .phi..sub.2 and .phi..sub.3, respectively, according to
States-TPPI (Cavanagh et al., Protein NMR Spetroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety). For acquisition of central peaks derived from
.sup.13C steady state magnetization, a second data set with
.phi..sub.1=-x is collected. The sum and the difference of the two
resulting data sets generate subspectra II and I, respectively,
containing the central peaks and peak pairs.
[0035] FIG. 3I illustrates the experimental scheme for the 3D
HCCH-TOCSY experiment. Rectangular 90.degree. and 180.degree.
pulses are indicated by thin and thick vertical bars, respectively,
and phases are indicated above the pulses. Where no radio-frequency
(r.f.) phase is marked, the pulse is applied along x. In order to
accomplish sine modulation, .phi..sub.1=y. The scaling factor
.kappa. for .sup.1H chemical shift evolution during t.sub.1 is set
to 1.0. The high power 90.degree. pulse lengths were: 5.8 .mu.s for
.sup.1H and 15.4 .mu.s for .sup.13C, and 38 .mu.s for .sup.15N.
.sup.13C decoupling during t.sub.1(.sup.1H) is achieved using a
(90.sub.x-180.sub.y-90.sub.x) composite pulse. The lengths of the
.sup.1H spin-lock purge pulses are: first SL.sub.x, 5.7 ms; second
SL.sub.x, 0.9 ms; SL.sub.y, 4.3 ms. SEDUCE is used for decoupling
of .sup.13CO during t.sub.1 and t.sub.2 (r.f. field strength=1
kHz), and GARP is employed for decoupling of .sup.13C during
acquisition (r.f.=2.5 kHz). The .sup.1H r.f carrier is placed at
the position of the solvent line at 0 ppm before the start of the
first semi constant time .sup.1H evolution period, and then
switched to the water line at 4.78 ppm after the second 90.degree.
.sup.1H pulse. The .sup.13C.sup..alpha. and .sup.15N r.f. carriers
are set to 38 ppm and 120.9 ppm, respectively. The length of
.sup.13C spin-lock purge pulses denoted SL.sub.x are of 2 ms
duration. .sup.13C isotropic mixing is accomplished using the
DIPSI-2 scheme (r.f.=8.5 kHz). The duration and strengths of the
pulsed z-field gradients (PFGs) are: G1 (100 .mu.s, 16 G/cm); G2 (2
ms, 15 G/cm); G3 (300 .mu.s, 8 G/cm); G4 (500 .mu.s, 30 G/cm); G5
(100 .mu.s, 16 G/cm). All PFG pulses are of rectangular shape. A
recovery delay of at least 100 .mu.s duration is inserted between a
PFG pulse and an r.f. pulse. The delays are: .tau..sub.1=850 .mu.s,
.tau..sub.2=3.2 ms. .sup.1H-frequency labeling in t.sub.1 is
achieved in a semi constant-time fashion with t.sub.1.sup.a(0)=1.7
ms, t.sub.1.sup.b(0)=1 .mu.s, t.sub.1.sup.c(0)=1.701 ms,
.DELTA.t.sub.1.sup.a=33.3 .mu.s, .DELTA.t.sub.1.sup.b=19.3 .mu.s,
.DELTA.t.sub.1.sup.c=-14 .mu.s. .sup.13C-frequency labeling in
t.sub.2 is achieved in a semi constant-time fashion with
t.sub.2.sup.a(0)=1120 .mu.s, t.sub.2.sup.b(0)=62.5 .mu.s,
t.sub.2.sup.c(0)=995 .mu.s, .DELTA.t.sub.2.sup.a=160 .mu.s,
.DELTA.t.sub.2.sup.b=125 .mu.s, .DELTA.t.sub.2.sup.c=-35 .mu.s.
These delays ensure that a 90.degree. first-order phase correction
is obtained along .omega..sub.2(.sup.13C). The fractional increases
of the semi constant-time period in t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.2.sup.c/.DELTA.t.sub.2.sup.a=0.58, and in
t.sub.2 equals to
.lamda.=1+.DELTA.t.sub.2.sup.c/.DELTA.t.sub.2.sup.a=0.78. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x, -x; .phi..sub.3=x;
.phi..sub.4=2(x), 2(-x); .phi..sub.5(receiver)=x, -x. Quadrature
detection in t.sub.1(.sup.13C) and t.sub.2(.sup.13C) is
accomplished by altering the phases .phi..sub.2 and .phi..sub.3,
respectively, according to States-TPPI (Cavanagh et al., Protein
NMR Spetroscopy, Academic Press, San Diego, (1996), which is hereby
incorporated by reference in its entirety). For acquisition of
central peaks derived from .sup.13C steady state magnetization, a
second data set with .phi..sub.1=-x is collected. The sum and the
difference of the two resulting data sets generate subspectra II
and I, respectively, containing the central peaks and peak
pairs.
[0036] FIG. 3J illustrates the experimental scheme for the 2D
HBCB(CGCD)HD experiment. Rectangular 90.degree. and 180.degree.
pulses are indicated by thin and thick vertical bars, respectively,
and phases are indicated above the pulses. Where no radio-frequency
(r.f.) phase is marked, the pulse is applied along x. In order to
accomplish sine modulation, .phi..sub.1=y. The scaling factor
.kappa. for .sup.1H chemical shift evolution during t.sub.1 is set
to 1.0. The high power 90.degree. pulse lengths were: 5.8 .mu.s for
.sup.1H and 15.4 .mu.s for .sup.13C. The first 180.degree. pulse on
.sup.13C prior to t.sub.1(.sup.13C) is applied at high power.
Subsequently, the 90.degree. pulse lengths of .sup.13C.sup..beta.
is adjusted to 66 .mu.s. The 180.degree. .sup.13C.sup..beta. and
.sup.13C.sup.aro pulses are of gaussian-3 shape and 375 .mu.s
duration. WALTZ16 is used for decoupling of .sup.1H (r.f. field
strength=4.5 kHz) during the magnetization transfer from
.sup.13C.sup..alpha. to .sup.13C.sup.aro, and GARP is employed to
decouple .sup.13C.sup.aro (r.f.=2.5 kHz) during acquisition. The
.sup.1H r.f. carrier is placed at 0 ppm before the start of the
semi constant time .sup.1H evolution period, and then switched to
the water line at 4.78 ppm after the second 90.degree. .sup.1H
pulse. The .sup.13C r.f. carrier is set to 38 ppm during
.omega..sub.1(.sup.13C.sup..beta.) and then switched to 131 ppm
before the first 90.degree. pulse on .sup.13C.sup.aro (pulse
labeled with .phi..sub.4). The duration and strengths of the pulsed
z-field gradients (PFGs) are: G1 (500 .mu.s, 2 G/cm); G2 (1 ms, 22
G/cm); G3 (2 ms, 10 G/cm); G4 (1 ms, 5 G/cm); G5 (500 .mu.s, 4
G/cm); G6 (1 ms, -14 G/cm); G7 (500 .mu.s, -2 G/cm). All PFG pulses
are of rectangular shape. A recovery delay of at least 100 .mu.s
duration is inserted between a PFG pulse and an r.f. pulse. The
delays are: .tau..sub.1=1.8 ms, .tau..sub.2=8.8 ms, .tau..sub.3=71
.mu.s, .tau..sub.4=5.4 ms, .tau..sub.5=4.2 ms, .tau..sub.6=710
.mu.s, .tau..sub.7=2.5 ms. .sup.1H-frequency labeling is achieved
in a semi constant-time fashion with t.sub.1.sup.a(0) 1.7 ms,
t.sub.1.sup.b(0)=1 .mu.s, t.sub.1.sup.c(0)=1.701 ms,
.DELTA.t.sub.1.sup.a=33.3 .mu.s, .DELTA.t.sub.1.sup.b=19.3 .mu.s,
.DELTA.t.sub.1.sup.c=-14 .mu.s. Hence, the fractional increase of
the semi constant-time period with t.sub.1 equals to
.lamda.=1+.DELTA.t.sub.1.sup.c/.DELTA.t.sub.1.sup.a, 0.58. Phase
cycling: .phi..sub.1=x; .phi..sub.2=x; .phi..sub.3=x, y, -x, -y;
.phi..sub.4=4(x), 4(-x); .phi..sub.5 (receiver) x, -x, x, -x, -x,
x, -x, x. Quadrature detection in t.sub.1(.sup.13C) is accomplished
by altering the phases .phi..sub.2 respectively, according to
States-TPPI. For acquisition of central peaks derived from .sup.13C
steady state magnetization, a second data set with .phi..sub.1=-x
is collected. The sum and the difference of the two resulting data
sets generate subspectra II and I, respectively, containing the
central peaks and peak pairs.
[0037] FIG. 3K illustrates the experimental scheme for the 2D
.sup.1H-TOCSY-relayed-HCH-COSY experiment. Rectangular 90.degree.
and 180.degree. pulses are indicated by thin and thick vertical
bars, respectively, and phases are indicated above the pulses.
Where no radio-frequency (r.f.) phase is marked, the pulse is
applied along x. In order to accomplish sine modulation,
.phi..sub.6=y. The high-power 90.degree. pulse lengths were: 5.9
.mu.s for .sup.1H and 15.4 .mu.s for .sup.13C. The .sup.1H r.f.
carrier is placed at the position of the solvent line at 4.78 ppm,
and the .sup.13C carrier is set to 131 ppm. GARP is used for
.sup.13C decoupling during acquisition (r.f. field strength 2.5
kHz), and .sup.1H isotropic mixing is accomplished using the
DIPSI-2 scheme (r.f.=16 kHz). The duration and strengths of the
pulsed z-field gradients (PFGs) are: G1 (1 ms, -10 G/cm); G2 (500
.mu.s, 6 G/cm); G3 (500 .mu.s, 7.5 G/cm); G4 (1 ms, 22 G/cm). All
PFG pulses are of rectangular shape. A recovery delay of at least
100 .mu.s duration is inserted between a PFG pulse and an r.f.
pulse. The delays are: .tau..sub.1=3.0 ms, .tau..sub.2=15.38 ms.
Phase cycling: .phi..sub.1x, -x; .phi..sub.2=x, x, y, y, -x, -x,
-y, -y; .phi..sub.3=4(x), 4(y), 4(-x), 4(-y); .phi..sub.4=x, x, -x,
-x; .phi..sub.5(receiver) x, -x, x, -x, -x, x, -x, x. Quadrature
detection in t.sub.1(.sup.13C) is accomplished by altering the
phase .phi..sub.1 according to States-TPPI.
[0038] FIG. 4 illustrates the polypeptide chemical shifts
correlated by the various spectra constituting the TR NMR
experiments identified for efficient HTP resonance assignment of
proteins. The nuclei for which the chemical shifts are obtained
from a given experiment are boxed and labeled accordingly.
[0039] FIG. 5 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup.N)]-strips
taken from 3D HACA(CO)NHN spectrum obtained for Z-domain protein
with a total measurement time of 6.7 hours. The 3D NMR data were
acquired with 40(t.sub.1).times.28(t.sub.2).times.512(t.sub.3)
complex points and t.sub.1max(.sup.13C.sup..alpha.;
.sup.1H.sup..alpha.)=6.5 ms, t.sub.2max(.sup.15N)=20.8 ms, and
t.sub.3max(.sup.1H.sup.N)=73.2 ms. Data acquisition and processing
were performed as shown in FIG. 1. The central peaks were acquired
from steady state .sup.13C magnetization. The strip labeled
"central peak" was taken from the 3D NMR spectrum comprising the
central peaks. The strip labeled (A) was taken from the 3D NMR
spectrum comprising in-phase peak pairs arising from cosine
modulation; strip (B) from the 3D NMR spectrum comprising
anti-phase peak pairs arising from sine modulation (the boxed peak
shows opposite peak intensity compared to the others); strip (C)
from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the sum of the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum, and strip (D) from the 3D
NMR spectrum comprising the edited, phase-sensitive peak generated
from the difference between the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum. All strips have been taken,
respectively, at the .sup.15N chemical shift (113.8 ppm) of Asp 50
along .omega..sub.2(.sup.15N) and centered about the corresponding
amide proton shifts detected along .omega..sub.3(.sup.1H.sup.N).
.sup.1H, .sup.13C, and .sup.15N chemical shifts are in ppm relative
to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS).
[0040] FIG. 6 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup.N)]-strips
taken from 3D HNNCAHA spectrum obtained for Z-domain protein with a
total measurement time of 10.7 hours. The 3D NMR data were acquired
with 64(t.sub.1).times.28(t.sub.2).times.512(t.sub.3) complex
points and t.sub.1max(.sup.13C.sup..alpha.;
.sup.1H.sup..alpha.)=12.1 ms, t.sub.2max(.sup.15N)=20.8 ms, and
t.sub.3max(.sup.1H.sup.N)=73.2 ms. Data acquisition and processing
were performed as shown in FIG. 1. The central peaks were acquired
from incomplete INEPT transfer of the .sup.13C magnetization. The
strip labeled "central peak" was taken from the 3D NMR spectrum
comprising the central peaks. The strip labeled (A) was taken from
the 3D NMR spectrum comprising in-phase peak pairs arising from
cosine modulation; strip (B) from the 3D NMR spectrum comprising
anti-phase peak pairs arising from sine modulation (the boxed peak
shows opposite peak intensity compared to the others); strip (C)
from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the sum of the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum and strip (D) from the 3D
NMR spectrum comprising the edited, phase-sensitive peak generated
from the difference between the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum. All strips have been taken,
respectively, at the .sup.15N chemical shift (117.4 ppm) of Leu 35
along .omega..sub.2(.sup.15N) and centered about the corresponding
amide proton shifts detected along .omega..sub.3(.sup.1H.sup.N).
.sup.1H, .sup.13C, and .sup.15N chemical shifts are in ppm relative
to DSS.
[0041] FIG. 7 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup.N)]-strips
taken from 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN
spectrum obtained for Z-domain protein with a total measurement
time of 12.7 hours. The 3D NMR data were acquired with
76(t.sub.1).times.28(t.sub.2).times.512(t.sub.3) complex points and
t.sub.1max(.sup.13C.sup..alpha./.beta.; .sup.1H.sup..alpha./.beta.)
6.25 ms, t.sub.2max(.sup.15N)=20.8 ms, and
t.sub.3max(.sup.1H.sup.N)=73.2 ms. Data acquisition and processing
were performed as shown in FIG. 1. The central peaks were acquired
from steady state .sup.13C magnetization. The strip labeled
"central peaks" was taken from the 3D NMR spectrum comprising the
central peaks. The strip labeled (A) was taken from the 3D NMR
spectrum comprising in-phase peak pairs arising from cosine
modulation; strip (B) from the 3D NMR spectrum comprising
anti-phase peak pairs arising from sine modulation (the boxed peaks
show opposite peak intensity compared to the others); strip (C)
from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the sum of the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum and strip (D) from the 3D
NMR spectrum comprising the edited, phase-sensitive peak generated
from the difference between the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum. All strips have been taken,
respectively, at the .sup.15N chemical shift (117.4 ppm) of Leu 35
along .omega..sub.2(.sup.15N) and centered about the corresponding
amide proton shifts detected along .omega..sub.3(.sup.1H.sup.N).
.sup.1H, .sup.13C, and .sup.15N chemical shifts are in ppm relative
to DSS.
[0042] FIG. 8 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup.N)]-strips
taken from 3D H.sup..alpha./.beta.C.sup..alpha./.beta.NHN spectrum
obtained for Z-domain protein with a total measurement time of 11.4
hours. The 3D NMR data were acquired with
68(t.sub.1).times.28(t.sub.2).times.512(t.sub.3) complex points and
t.sub.1max(.sup.13C.sup..alpha./.beta.;
.sup.1H.sup..alpha./.beta.)=5.6 ms, t.sub.2max(.sup.15N)=20.8 ms,
and t.sub.3max(.sup.1H.sup.N)=73.2 ms. Data acquisition and
processing were performed as shown in FIG. 1. The central peaks
were acquired from steady state .sup.13C magnetization. The strip
labeled "central peaks" was taken from the 3D NMR spectrum
comprising the central peaks. The strip labeled (A) was taken from
the 3D NMR spectrum comprising in-phase peak pairs arising from
cosine modulation; strip (B) from the 3D NMR spectrum comprising
anti-phase peak pairs arising from sine modulation (the boxed peaks
show opposite peak intensity compared to the others); strip (C)
from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the sum of the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum, and strip (D) from the 3D
NMR spectrum comprising the edited, phase-sensitive peak generated
from the difference between the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum. All strips have been taken,
respectively, at the .sup.15N chemical shift (117.4 ppm) of Leu 35
along .omega..sub.2(.sup.15N) and centered about the corresponding
amide proton shifts detected along .omega..sub.3(.sup.1H.sup.N).
.sup.1H, .sup.13C, and .sup.15N chemical shifts are in ppm relative
to DSS.
[0043] FIG. 9 shows a composite plot of
[.omega..sub.1(.sup.13C'/.sup.13C.sup..alpha.),.omega..sub.3(.sup.1H.sup.-
N]-strips taken from 3D HNN<CO,CA> spectrum obtained for
Z-domain protein with a total measurement time of 9.4 hours. The 3D
NMR data were acquired with
56(t.sub.1).times.28(t.sub.2).times.512(t.sub.3) complex points and
t.sub.1max(.sup.13C'; .sup.13C.sup..alpha.)=6.9 ms,
t.sub.2max(.sup.15N)=20.8 ms, and t.sub.3max(.sup.1H.sup.N)=73.2
ms. Data acquisition and processing were performed as shown in FIG.
1. The central peaks were acquired from steady state .sup.13C
magnetization. The strip labeled "central peak" was taken from the
3D NMR spectrum comprising the central peaks. The strip labeled (A)
was taken from the 3D NMR spectrum comprising in-phase peak pairs
arising from cosine modulation; strip (B) from the 3D NMR spectrum
comprising anti-phase peak pairs arising from sine modulation (the
boxed peaks show opposite peak intensity compared to the others);
strip (C) from the 3D NMR spectrum comprising the edited,
phase-sensitive peak generated from the sum of the cosine-modulated
3D NMR spectrum and the sine-modulated 3D NMR spectrum, and strip
(D) from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the difference between the cosine-modulated 3D
NMR spectrum and the sine-modulated 3D NMR spectrum. All strips
have been taken, respectively, at the .sup.15N chemical shift
(170.0 ppm) of Phe 43 along .omega..sub.2(.sup.15N) and centered
about the corresponding amide proton shifts detected along
.omega..sub.3(.sup.1H.sup.N). .sup.1H, .sup.13C, and .sup.15N
chemical shifts are in ppm relative to DSS.
[0044] FIG. 10 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup.N)]-strips
taken from 3D HC(C-TOCSY-CO)NHN spectrum obtained for Z-domain
protein with a total measurement time of 12.7 hours. The 3D NMR
data were acquired with
76(t.sub.1).times.28(t.sub.2).times.512(t.sub.3) complex points and
t.sub.1max(.sup.13C; .sup.1H)=6.3 ms, t.sub.2max(.sup.15N)=20.8 ms,
and t.sub.3max(.sup.1H.sup.N)=73.2 ms. Data acquisition and
processing were performed as shown in FIG. 1. The central peaks
were acquired from steady state .sup.13C magnetization. The strip
labeled "central peaks" was taken from the 3D NMR spectrum
comprising the central peaks. The strip labeled (A) was taken from
the 3D NMR spectrum comprising in-phase peak pairs arising from
cosine modulation; strip (B) from the 3D NMR spectrum comprising
anti-phase peak pairs arising from sine modulation (the boxed peaks
show opposite peak intensity compared to the others); strip (C)
from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the sum of the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum and strip (D) from the 3D
NMR spectrum comprising the edited, phase-sensitive peak generated
from the difference between the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum. All strips have been taken,
respectively, at the .sup.15N chemical shift (118.8 ppm) of Phe 18
along .omega..sub.2(.sup.15N) and centered about the corresponding
amide proton shifts detected along .omega..sub.3(.sup.1H.sup.N).
.sup.1H, .sup.13C, and .sup.15N chemical shifts are in ppm relative
to DSS.
[0045] FIG. 11 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup..beta.)]-strip-
s taken from 3D HCCH-COSY spectrum obtained for Z-domain protein
with a total measurement time of 10.8 hours. The 3D NMR data were
acquired with 90(t.sub.1).times.20(t.sub.2).times.512(t.sub.3)
complex points and t.sub.1max(.sup.13C; .sup.1H)=7.4 ms,
t.sub.2max(.sup.13C)=6.1 ms, and t.sub.3max(.sup.1H)=73.2 ms. Data
acquisition and processing were performed as shown in FIG. 1. The
central peaks were acquired from steady state .sup.13C
magnetization. The strip labeled "central peaks" was taken from the
3D NMR spectrum comprising the central peaks. The strip labeled (A)
was taken from the 3D NMR spectrum comprising in-phase peak pairs
arising from cosine modulation; strip (B) from the 3D NMR spectrum
comprising anti-phase peak pairs arising from sine modulation (the
boxed peaks show opposite peak intensity compared to the others);
strip (C) from the 3D NMR spectrum comprising the edited,
phase-sensitive peak generated from the sum of the cosine-modulated
3D NMR spectrum and the sine-modulated 3D NMR spectrum, and strip
(D) from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the difference between the cosine-modulated 3D
NMR spectrum and the sine-modulated 3D NMR spectrum. All strips
have been taken, respectively, at the .sup.13C.sup..beta. chemical
shift (37.8 ppm) of Asn 19 along .omega..sub.2(.sup.13C) and
centered about the corresponding proton (.sup.1H.sup..beta.) shifts
detected along .omega..sub.3(.sup.1H). .sup.1H and .sup.13C
chemical shifts are in ppm relative to DSS.
[0046] FIG. 12 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup..beta.)]-strip-
s taken from 3D HCCH-TOCSY spectrum obtained for Z-domain protein
with a total measurement time of 11.0 hours. The 3D NMR data were
acquired with 80(t.sub.1).times.20(t.sub.2).times.512(t.sub.3)
complex points and t.sub.1max(.sup.13C; .sup.1H)=6.6 ms,
t.sub.2max(.sup.13C)=6.1 ms, and t.sub.3max(.sup.1H)=73.2 ms. Data
acquisition and processing were performed as shown in FIG. 1. The
central peaks were acquired from steady state .sup.13C
magnetization. The strip labeled "central peaks" was taken from the
3D NMR spectrum comprising the central peaks. The strip labeled (A)
was taken from the 3D NMR spectrum comprising in-phase peak pairs
arising from cosine modulation; strip (B) from the 3D NMR spectrum
comprising anti-phase peak pairs arising from sine modulation (the
boxed peaks show opposite peak intensity compared to the others);
strip (C) from the 3D NMR spectrum comprising the edited,
phase-sensitive peak generated from the sum of the cosine-modulated
3D NMR spectrum and the sine-modulated 3D NMR spectrum, and strip
(D) from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the difference of the cosine-modulated 3D NMR
spectrum between the sine-modulated 3D NMR spectrum. All strips
have been taken, respectively, at the .sup.13C.sup..beta. chemical
shift (37.8 ppm) of Asn 36 along .omega..sub.2(.sup.13C) and
centered about the corresponding proton (.sup.1H.sup..beta.) shifts
detected along .omega..sub.3(.sup.1H). .sup.1H and .sup.13C
chemical shifts are in ppm relative to DSS.
[0047] FIG. 13 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup..delta.)]-stri-
ps taken from 2D HBCB(CGCD)HD spectrum obtained for Z-domain
protein with a total measurement time of 7.8 hours. The 2D NMR data
were acquired with 76(t.sub.1).times.512(t.sub.2) complex points
and t.sub.1max(.sup.13C; .sup.1H)=6.3 ms and
t.sub.2max(.sup.1H)=73.2 ms. Data acquisition and processing were
performed as shown in FIG. 1. The central peaks were acquired from
steady state .sup.13C magnetization. The strip labeled "central
peak" was taken from the 2D NMR spectrum comprising the central
peaks. The strip labeled (A) was taken from the 2D NMR spectrum
comprising in-phase peak pairs arising from cosine modulation;
strip (B) from the 2D NMR spectrum comprising anti-phase peak pairs
arising from sine modulation (the boxed peaks show opposite peak
intensity compared to the others); strip (C) from the 2D NMR
spectrum comprising the edited, phase-sensitive peak generated from
the sum of the cosine-modulated 2D NMR spectrum and the
sine-modulated 2D NMR spectrum, and strip (D) from the 2D NMR
spectrum comprising the edited, phase-sensitive peak generated from
the difference of the cosine-modulated 2D NMR spectrum between the
sine-modulated 2D NMR spectrum. All strips have been centered about
the aromatic proton (.sup.1H.sup..delta.) shifts of Phe 18 detected
along .omega..sub.2(.sup.1H). .sup.1H and .sup.13C chemical shifts
are in ppm relative to DSS.
[0048] FIG. 14 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H.sup..alpha.)]-stri-
ps taken from 3D H.sup..alpha./.beta.C.sup..alpha./.beta.COHA
spectrum obtained for ubiquitin protein with a total measurement
time of 12.7 hours. The 3D NMR data were acquired with
76(t.sub.1).times.32(t.sub.2).times.512(t.sub.3) complex points and
t.sub.1max(.sup.13C.sup..alpha./.beta.;
.sup.1H.sup..alpha./.beta.)=6.3 ms, t.sub.2max(.sup.13C')=17.2 ms,
and t.sub.3max(.sup.1H.sup..alpha.)=72.8 ms. Data acquisition and
processing were performed as shown in FIG. 1. The central peaks
were acquired from the steady state .sup.13C magnetization. The
strip labeled "central peaks" was taken from the 3D NMR spectrum
comprising the central peaks. The strip labeled (A) was taken from
the 3D NMR spectrum comprising in-phase peak pairs arising from
cosine modulation; strip (B) from the 3D NMR spectrum comprising
anti-phase peak pairs arising from sine modulation (the boxed peaks
show opposite peak intensity compared to the others); strip (C)
from the 3D NMR spectrum comprising the edited, phase-sensitive
peak generated from the sum of the cosine-modulated 3D NMR spectrum
and the sine-modulated 3D NMR spectrum, and strip (D) from the 3D
NMR spectrum comprising the edited, phase-sensitive peak generated
from the difference of the cosine-modulated 3D NMR spectrum between
the sine-modulated 3D NMR spectrum. All strips have been taken,
respectively, at the .sup.13C' chemical shift (175.2 ppm) of Pro 19
along .omega..sub.2(.sup.13C') and centered about the corresponding
proton shifts detected along .omega..sub.3(.sup.1H.sup..alpha.).
.sup.1H and .sup.13C chemical shifts are in ppm relative to
DSS.
[0049] FIG. 15 shows a composite plot of
[.omega..sub.1(.sup.13C/.sup.1H),.omega..sub.3(.sup.1H)]-strips
taken from 2D H-TOCSY-HCH-COSY spectrum obtained for ubiquitin
protein with a total measurement time of 1.4 hours. The 2D NMR data
were acquired with 100(t.sub.1).times.512(t.sub.2) complex points
and t.sub.1max(.sup.13C; .sup.1H)=9.9 ms and
t.sub.2max(.sup.1H)=72.8 ms. Data acquisition and processing were
performed as shown in FIG. 1. No central peaks were acquired. The
strip labeled (A) was taken from the 3D NMR spectrum comprising
in-phase peak pairs arising from cosine modulation; strip (B) from
the 3D NMR spectrum comprising anti-phase peak pairs arising from
sine modulation (the boxed peaks show opposite peak intensity
compared to the others); strip (C) from the 3D NMR spectrum
comprising the edited, phase-sensitive peak generated from the sum
of the cosine-modulated 3D NMR spectrum and the sine-modulated 3D
NMR spectrum, and strip (D) from the 3D NMR spectrum comprising the
edited, phase-sensitive peak generated from the difference of the
cosine-modulated 3D NMR spectrum between the sine-modulated 3D NMR
spectrum. All strips have been centered about the aromatic proton
shifts detected along .omega..sub.2(.sup.1H). .sup.1H and .sup.13C
chemical shifts are in ppm relative to DSS.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention discloses eleven RD TR NMR experiments
where the chemical shift values encoded in a peak pair of an NMR
spectrum are detected in a phase sensitive manner. The present
invention also discloses different combinations of those
phase-sensitively detected RD NMR experiments which allow one to
obtain sequential backbone chemical shift assignments for
determining the secondary structure of a protein molecule and
nearly complete assignments of chemical shift values for a protein
molecule including aliphatic and aromatic sidechain spin
systems.
[0051] The basic acquisition principle for recording edited, phase
sensitive RD NMR spectra is illustrated in FIG. 1 (Brutscher et
al., J. Magn Reson., B 109:238-242 (1995), which is hereby
incorporated by reference in its entirety). The joint evolution of
two chemical shifts, .OMEGA..sub.x and .OMEGA..sub.y, can result in
four different time domain NMR data sets, (1), (2), (3), and (4),
which are generated from all possible combinations of sine
modulation and cosine modulation with either of the two chemical
shifts (shown on the left-hand side of the equations for data sets
(1)-(4)). According to the addition theorems of trigonometric
functions, these products of sine and cosine functions are equal to
sums of sine and cosine functions of sums and differences of the
two chemical shifts (shown on the right-hand side of the equations
for data sets (1)-(4)). To obtain an absorptive NMR signal, two out
of the four data sets need to be considered as "real" and
"imaginary" parts for cosine-modulated RD NMR and sine-modulated RD
NMR. Data sets (1) and (2) correspond to real part and imaginary
part, respectively, of a cosine-modulated RD NMR experiment, in
which .OMEGA..sub.y is detected in a phase sensitive manner and the
evolution of the chemical shift .OMEGA..sub.x effects a cosine
modulation (see (A) in FIG. 1). Data sets (3) and (4) correspond to
real part and imaginary part, respectively, of a sine-modulated RD
NMR experiment, in which .OMEGA..sub.y is detected in a phase
sensitive manner and the evolution of .OMEGA..sub.x effects a sine
modulation, further enabling the phase sensitive detection of
.OMEGA..sub.x (see (B) in FIG. 1). Thus, the phase-sensitively
detected sine-modulated RD NMR spectrum contains all information
about .OMEGA..sub.x and .OMEGA..sub.y for unambiguous chemical
shift assignment. Appropriate linear combination of real and
imaginary parts of the cosine-modulated and the sine-modulated RD
NMR experiments yields the edited, phase sensitive RD NMR spectra,
which also contain all information about .OMEGA..sub.x and
.OMEGA..sub.y (see (C) and (D) in FIG. 1).
[0052] FIG. 2 provides a survey of (i) the names, (ii) the
magnetization transfer pathways and (iii) the peak patterns
observed in the projected dimension of specific embodiments of the
eleven RD NMR experiments disclosed by the present invention. For
simplicity, FIG. 2 shows the in-phase peak pattern from the
cosine-modulated RD NMR experiments. When acquiring the
sine-modulated congeners, anti-phase peak pairs are observed
instead (see (B) in FIG. 1). The group comprising the first three
experiments are designed to yield "sequential" connectivities via
one-bond scalar couplings: 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN (FIG. 2A), 3D
HACA(CO)NHN (FIG. 2B), and 3D HC(C-TOCSY-CO)NHN (FIG. 2C). The
following three experiments provide "intraresidual" connectivities
via one-bond scalar couplings: 3D HNNCAHA (FIG. 2D), 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.COHA (FIG. 2E), and 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.NHN (FIG. 2F). 3D
HNN<CO,CA> (FIG. 2G) offers both intraresidual
.sup.1H.sup.N-.sup.13C.sup..alpha. and sequential
.sup.1H.sup.N--.sup.13C.sup..alpha. connectivities. Although 3D
HNNCAHA (FIG. 2D), 3D H.sup..alpha./.beta.C.sup..alpha./.beta.
(FIG. 2F) and 3D HNN<CO,CA> (FIG. 2G) also provide sequential
connectivities via two-bond
.sup.13C.sup..alpha..sub.i-1-.sup.15N.sub.i scalar couplings, those
are usually smaller than the one-bond couplings (Cavanagh et al.,
Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which
is hereby incorporated by reference in its entirety), and obtaining
complete backbone resonance assignments critically depends on
experiments designed to provide sequential connectivities via
one-bond couplings (FIGS. 2D-F). 3D HCCH-COSY (FIG. 2H) and 3D
HCCH-TOCSY (FIG. 2I) allow one to obtain assignments for the
"aliphatic" side chain spin systems, while 2D HBCB(CDCG)HD (FIG.
2J) and 2D .sup.1H-TOCSY-relayed HCH-COSY (FIG. 2K) provide the
corresponding information for the "aromatic" spin systems.
[0053] The RD NMR experiments are grouped accordingly in Table 1,
which lists for each experiment (i) the nuclei for which the
chemical shifts are measured, (ii) if and how the central peaks are
acquired and (iii) additional notable technical features.
State-of-the art implementations (Cavanagh et al., Protein NMR
Spectroscopy, Academic Press, San Diego, (1996); Kay, J. Am. Chem.
Soc., 115:2055-2057 (1993); Grzesiek et al., J. Magn Reson.,
99:201-207 (1992); Montelione et al., J. Am. Chem. Soc.,
114:10974-10975 (1992); Boucher et al., J. Biomol. NMR, 2:631-637
(1992); Yamazaki et al., J. Am. Chem. Soc., 115:11054-11055 (1993);
Zerbe et al., J. Biomol. NMR, 7:99-106 (1996); Grzesiek et al., J.
Biomol. NMR, 3:185-204 (1993), which are hereby incorporated by
reference in their entirety) making use of pulsed field z-gradients
for coherence selection and/or rejection, and sensitivity
enhancement (Cavanagh et al., Protein NMR Spectroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) were chosen, which allow executing these
experiments with a single transient per acquired free induction
decay (FID). Semi (Grzesiek et al., J. Biomol. NMR, 3:185-204
(1993), which is hereby incorporated by reference in its entirety)
constant-time (Cavanagh et al., Protein NMR Spectroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) chemical shift frequency-labeling modules were
used throughout in the indirect dimensions in order to minimize
losses arising from transverse nuclear spin relaxation. FIGS. 3A-K
provide comprehensive descriptions of the RD NMR r.f. pulse
sequences used in the eleven RD NMR experiments.
TABLE-US-00001 TABLE 1 Phase-Sensitively Detected Reduced
Dimensionality NMR Experiments Experiment Nuclei for which the
chemical shifts are Acquisition of (see FIG. 2) correlated.sup.a,b
central peaks.sup.c 3D spectra for sequential backbone
connectivities: (A)
H.sup..alpha./.sup..beta.C.sup..alpha./.sup..beta.(CO)NHN
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1,
.sup.1H.sup..alpha..sub.i-1, .sup.13C.sup..alpha..sub.i-1,
.sup.15N.sub.i, .sup.1H.sup.N.sub.i .sup.13C (B) HACA(CO)NHN
.sup.1H.sup..alpha..sub.i-1, .sup.13C.sup..alpha..sub.i-1,
.sup.15N.sub.i, .sup.1H.sup.N.sub.i .sup.13C (C) HC(C-TOCSY--CO)NHN
.sup.1H.sup.a1i.sub.i-1, .sup.13C.sup.ali.sub.i-1, .sup.15N.sub.i,
.sup.1H.sup.N.sub.i .sup.13C 3D spectra for intraresidual backbone
connectivities: (D) HNNCAHA.sup.b,d .sup.1H.sup..alpha..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, .sup.1H.sup.N.sub.i
INEPT (E) H.sup..alpha./bC.sup..alpha./bCOHA
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.13C.dbd.O.sub.i .sup.13C (F) H.sup..alpha./bC.sup..alpha./bNH
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, .sup.1H.sup.N.sub.i .sup.13C 3D spectrum for intra-
and sequential backbone connectivities: (G) HNN<CO,CA>.sup.b
.sup.13C.dbd.O.sub.i.sub.-1, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, .sup.1H.sup.N.sub.i INEPT 3D spectra for assignment
of aliphatic resonances:.sup.e (H) HCCH--COSY .sup.1H.sub.m,
.sup.13C.sub.m, .sup.1H.sub.n, .sup.13C.sub.n .sup.13C (I)
HCCH-TOCSY .sup.1H.sub.m, .sup.13C.sub.m, .sup.1H.sub.n,
.sup.13C.sub.n, .sup.1H.sub.p, .sup.13C.sub.p .sup.13C 2D spectra
for assignment of aromatic resonances:.sup.e (J) HBCB(CGCD)HD
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..delta.
.sup.13C (K) .sup.1H-TOCSY--HCH--COSY .sup.1H.sub.m,
.sup.13C.sub.m, .sup.1H.sub.n, none.sup.f .sup.ai-1, i: numbers of
two sequentially located amino acid residues. .sup.bSequential
connectivities via two-bond
.sup.13C.sup..alpha..sub.i-1-.sup.15N.sub.i scalar couplings are
not considered in this table. .sup.capproach-1 (Szyperski et al.,
J. Am. Chem. Soc., 118: 8146-8147 (1996), which is hereby
incorporated by reference in its entirety): use of incomplete INEPT
(rows labeled with "INEPT"); approach-2 (Szyperski et al., J. Am.
Chem. Soc., 118: 8146-8147 (1996), which is hereby incorporated by
reference in its entirety): use of .sup.13C steady state
magnetization (rows labeled with ".sup.13C"). .sup.dadiabatic
.sup.13C.sup..beta.-decoupling (Kupce et al., J. Magn. Reson., A
115: 273-277 (1995); Matsuo et al., J. Magn. Reson. B 113: 190-194
(1996), which are hereby incorporated by reference in their
entirety) is employed during delays with transverse
.sup.13C.sup..alpha. magnetization. .sup.em, n, p: atom numbers in
neighboring CH, CH.sub.2 or CH.sub.3 groups. .sup.facquisition of
central peaks is prevented by the use of spin-lock purge pulses
(flanking the total correlation relay) to obtain pure phases.
The 3D HA,CA,(CO),N,HN Experiment
[0054] The present invention relates to a method of conducting a
reduced dimensionality three-dimensional (3D) HA,CA,(CO),N,HN
nuclear magnetic resonance (NMR) experiment by measuring the
chemical shift values for the following nuclei of a protein
molecule having two consecutive amino acid residues, i-1 and i: (1)
an .alpha.-proton of amino acid residue i-1,
.sup.1H.sup..alpha..sub.i-1; (2) an .alpha.-carbon of amino acid
residue i-1, .sup.13C.sup..alpha..sub.i-1; (3) a polypeptide
backbone amide nitrogen of amino acid residue i, .sup.15N.sub.i;
and (4) a polypeptide backbone amide proton of amino acid residue
i, .sup.1H.sup.N.sub.i, where the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 which
are encoded in a peak pair of a 3D NMR spectrum are detected in a
phase sensitive manner. The method involves providing a protein
sample and applying radiofrequency pulses to the protein sample
which effect a nuclear spin polarization transfer where the
chemical shift evolutions of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 of amino acid residue i-1 are
connected to the chemical shift evolutions of .sup.15N.sub.i and
.sup.1H.sup.N.sub.i of amino acid residue i, under conditions
effective (1) to generate NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i-1 and .sup.15N.sub.i in a
phase sensitive manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup..alpha.) and t.sub.2(.sup.15N), respectively,
and the chemical shift value of .sup.1H.sup.N.sub.i in a direct
time domain dimension, t.sub.3(.sup.1H.sup.N), and (2) to sine
modulate the .sup.13C.sup..alpha..sub.i-1 chemical shift evolution
in t.sub.1(.sup.13C.sup..alpha.) with the chemical shift evolution
of .sup.1H.sup..alpha..sub.i-1. Then, the NMR signals are processed
to generate a sine-modulated 3D NMR spectrum with an anti-phase
peak pair derived from the sine modulating, where (1) the chemical
shift values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i are measured
in two frequency domain dimensions, .omega..sub.2(.sup.15N) and
.omega..sub.3(.sup.1H.sup.N), respectively, and (2) the chemical
shift values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha.), by the frequency
difference between the two peaks forming the anti-phase peak pair
and the frequency at the center of the two peaks, respectively,
where the sine-modulated 3D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sup..alpha..sub.i-1 in a phase
sensitive manner.
[0055] In addition, the method of conducting a RD 3D
HA,CA,(CO),N,HN NMR experiment can involve applying radiofrequency
pulses under conditions effective (1) to generate additional NMR
signals encoding the chemical shift values of
.sup.13C.sup..alpha..sub.i-1 and .sup.15N.sub.i in a phase
sensitive manner in t.sub.1(.sup.13C.sup..alpha.) and
t.sub.2(.sup.15N), respectively, and the chemical shift value of
.sup.1H.sup.N.sub.i in t.sub.3(.sup.1H.sup.N) and (2) to cosine
modulate the .sup.13C.sup..alpha..sub.i-1 chemical shift evolution
in t.sub.1(.sup.13C.sup..alpha.) with the chemical shift evolution
of .sup.1H.sup..alpha..sub.i-1 for the additional NMR signals.
Then, the NMR signals and the additional NMR signals are processed
to further generate a cosine-modulated 3D NMR spectrum with an
in-phase peak pair derived from the cosine modulating, a sum 3D NMR
spectrum generated by adding the sine-modulated 3D NMR spectrum and
the cosine-modulated 3D NMR spectrum and a difference 3D NMR
spectrum generated by subtracting the cosine-modulated 3D NMR
spectrum from the sine-modulated 3D NMR spectrum. The combined use
of the sum 3D NMR spectrum and the difference 3D NMR spectrum
enables placement of the two peaks forming the peak pairs into
separate spectra, thereby allowing phase-sensitive editing of the
two peaks forming the peak pairs.
[0056] In addition, the method of conducting a RD 3D
HA,CA,(CO),N,HN NMR experiment can involve applying radiofrequency
pulses under conditions effective (1) to generate additional NMR
signals encoding the chemical shift values of
.sup.13C.sup..alpha..sub.i-1 and .sup.15N.sub.i in a phase
sensitive manner in t.sub.1(.sup.13C.sup..alpha.) and
t.sub.2(.sup.15N) and the chemical shift value of
.sup.1H.sup.N.sub.i in t.sub.3(.sup.1H.sup.N), and (2) to avoid
sine modulating the .sup.13C.sup..alpha..sub.i-1 chemical shift
evolution in t.sub.1(.sup.13C.sup..alpha.) with the chemical shift
evolution of .sup.1H.sup..alpha..sub.i-1 for the additional NMR
signals. Then, the NMR signals and the additional NMR signals are
processed to generate a 3D NMR spectrum with an additional peak
located centrally between two peaks forming the primary peak pair
which measures the chemical shift value of
.sup.13C.sup..alpha..sub.i-1 along
.omega..sub.1(.sup.13C.sup..alpha.). That additional peak can be
derived from .sup.13C.sup..alpha. nuclear spin polarization. One
specific embodiment (3D HACA(CO)NHN) of this method is illustrated
in FIG. 2B, where the applying radiofrequency pulses effects a
nuclear spin polarization transfer where a radiofrequency pulse is
used to create transverse .sup.1H.sup..alpha..sub.i-1
magnetization, which is transferred to
.sup.13C.sup..alpha..sub.i-1, to .sup.15N.sub.i, and to
.sup.1H.sup.N.sub.i, to generate the NMR signal. Another specific
embodiment of this method involves applying radiofrequency pulses
by (1) applying a first set of radiofrequency pulses according to
the scheme shown in FIG. 3B to generate a first NMR signal, and (2)
applying a second set of radiofrequency pulses according to the
scheme shown in FIG. 3B, where phase .phi..sub.1 of the first
.sup.1H pulse is altered by 180.degree. to generate a second NMR
signal. Then, prior to the processing, the first NMR signal and the
second NMR signal are added and subtracted whereby the NMR signals
are processed to generate a first NMR subspectrum derived from the
subtracting which contains the primary peak pair and a second NMR
subspectrum derived from the adding which contains the additional
peak located centrally between the two peaks forming the primary
peak pair.
[0057] In an alternate embodiment, the RD 3D HA,CA,(CO),N,HN NMR
experiment can be modified to a RD2D HA,CA,(CO,N),HN NMR experiment
which involves applying radiofrequency pulses so that the chemical
shift evolution of .sup.15N.sub.i does not occur. Then, the NMR
signals are processed to generate a two dimensional (2D) NMR
spectrum with a peak pair where (1) the chemical shift value of
.sup.1H.sup.N.sub.i is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.N), and (2) the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha.), by the frequency difference
between the two peaks forming the peak pair and the frequency at
the center of the two peaks, respectively.
[0058] In an alternate embodiment, the RD 3D HA,CA,(CO),N,HN NMR
experiment can be modified to a RD 4D HA,CA,CO,N,HN NMR experiment
which involves applying radiofrequency pulses so that the chemical
shift evolution of a polypeptide backbone carbonyl carbon of amino
acid residue i-1, .sup.13C'.sub.i-1, occurs under conditions
effective to generate NMR signals encoding the chemical shift value
of .sup.13C'.sub.i-1 in a phase sensitive manner in an indirect
time domain dimension, t.sub.4(.sup.13C'). Then, the NMR signals
are processed to generate a four dimensional (4D) NMR spectrum with
a peak pair where (1) the chemical shift values of .sup.15N.sub.i,
.sup.1H.sup.N.sub.i and .sup.13C'.sub.i-1 are measured in three
frequency domain dimensions, .omega..sub.2(.sup.15N),
.omega..sub.3(.sup.1H.sup.N) and .omega..sub.4(.sup.13C'),
respectively, and (2) the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha.), by the frequency difference
between the two peaks forming the peak pair and the frequency at
the center of the two peaks, respectively.
The 3D H,C,(C-TOCSY-CO),N,HN Experiment
[0059] The present invention also relates to a method of conducting
a reduced dimensionality three-dimensional (3D)
H,C,(C-TOCSY-CO),N,HN nuclear magnetic resonance (NMR) experiment
by measuring the chemical shift values for the following nuclei of
a protein molecule having two consecutive amino acid residues, i-1
and i: (1) aliphatic protons of amino acid residue i-1,
.sup.1H.sup.ali.sub.i-1; (2) aliphatic carbons of amino acid
residue i-1, .sup.13C.sup.ali.sub.i-1; (3) a polypeptide backbone
amide nitrogen of amino acid residue i, .sup.15N.sub.i; and (4) a
polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i, where the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 of amino acid residue i-1 are connected to
the chemical shift evolutions of .sup.15N.sub.i and
.sup.1H.sup.N.sub.i of amino acid residue i, under conditions
effective (1) to generate a NMR signal encoding the chemical shifts
of .sup.13C.sup.ali.sub.i-1 and .sup.15N.sub.i in a phase sensitive
manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup.ali) and t.sub.2(.sup.15N), respectively, and
the chemical shift of .sup.1H.sup.N.sub.i in a direct time domain
dimension, t.sub.3(.sup.1H.sup.N), and (2) to sine modulate the
chemical shift evolutions of .sup.13C.sup.ali.sub.i-1 in
t.sub.1(.sup.13C.sup.ali) with the chemical shift evolutions of
.sup.1H.sup.ali.sub.i-1. Then, the NMR signals are processed to
generate a sine-modulated 3D NMR spectrum with anti-phase peak
pairs derived from the sine modulating where (1) the chemical shift
values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i are measured in
two frequency domain dimensions, .omega..sub.2(.sup.15N) and
.omega..sub.3(.sup.1H.sup.N), respectively, and (2) the chemical
shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.ali), by the frequency
differences between each of the two peaks forming each of the
anti-phase peak pairs and the frequencies at the center of the two
peaks, respectively, where the sine-modulated 3D NMR spectrum
enables detection of the chemical shift value of
.sup.1H.sup.ali.sub.i-1 in a phase sensitive manner.
[0060] In addition, the method of conducting a RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment can involve applying
radiofrequency pulses under conditions effective (1) to generate
additional NMR signals encoding the chemical shift values of
.sup.13C.sup.ali.sub.i-1 and .sup.15N.sub.i in a phase sensitive
manner in t.sub.1(.sup.13C.sup.ali) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i
in t.sub.3(.sup.1H.sup.N) and (2) to cosine modulate the chemical
shift evolutions of .sup.13C.sup.ali.sub.i-1 in
t.sub.1(.sup.13C.sup.ali) with the chemical shift evolutions of
.sup.1H.sup.ali.sub.i-1 for the additional NMR signals. Then, the
NMR signals and the additional NMR signals are processed to further
generate a cosine-modulated 3D NMR spectrum with in-phase peak
pairs derived from the cosine modulating, a sum 3D NMR spectrum
generated by adding the sine-modulated 3D NMR spectrum and the
cosine-modulated 3D NMR spectrum and a difference 3D NMR spectrum
generated by subtracting the cosine-modulated 3D NMR spectrum from
the sine-modulated 3D NMR spectrum. The combined use of the sum 3D
NMR spectrum and the difference 3D NMR spectrum enables placement
of the two peaks forming the peak pairs into separate spectra,
thereby allowing phase-sensitive editing of the two peaks forming
the peak pairs.
[0061] In addition, the method of conducting a RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment can involve applying
radiofrequency pulses under conditions effective (1) to generate
additional NMR signals encoding the chemical shift values of
.sup.13C.sup.ali.sub.i-1 and .sup.15N.sub.i in a phase sensitive
manner in t.sub.1(.sup.13C.sup.ali) and t.sub.2(.sup.15N) and the
chemical shift value of .sup.1H.sup.N.sub.i in
t.sub.3(.sup.1H.sup.N), and (2) to avoid sine modulating the
chemical shift evolutions of .sup.13C.sup.ali.sub.i-1 in
t.sub.1(.sup.13C.sup.ali) with the chemical shift evolution of
.sup.1H.sup..alpha..sub.i-1 for the additional NMR signals. Then,
the NMR signals and the additional NMR signals are processed to
generate a 3D NMR spectrum with additional peaks located centrally
between two peaks forming the peak pairs which measure the chemical
shift values of .sup.13C.sup.ali.sub.i-1 along
.omega..sub.1(.sup.13C.sup.ali). Those additional peaks can be
derived from .sup.13C.sup.ali nuclear spin polarization. One
specific embodiment (3D HC-(C-TOCSY-CO)NHN) of this method is
illustrated in FIG. 2C, where the applying radiofrequency pulses
effects a nuclear spin polarization transfer, where a
radiofrequency pulse is used to create transverse
.sup.1H.sup.ali.sub.i-1 magnetization, and .sup.1H.sup.ali.sub.i-1
magnetization is transferred to .sup.13C.sup.ali.sub.i-1, to
.sup.13C.sup..alpha..sub.i-1, to .sup.13C'.sub.i-1, to
.sup.15N.sub.i, and to .sup.1H.sup.N.sub.i, where the NMR signal is
detected. Another specific embodiment of this method involves
applying radiofrequency pulses by (1) applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3C to
generate a first NMR signal, and (2) applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3C,
where phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal. Then, prior to the
processing, the first NMR signal and the second NMR signal are
added and subtracted, whereby the NMR signals are processed to
generate a first NMR subspectrum derived from the subtracting which
contains the peak pairs, and a second NMR subspectrum derived from
the adding which contains the additional peaks located centrally
between the two peaks forming the peak pairs.
[0062] In an alternate embodiment, the RD 3D H,C,(C-TOCSY-CO),N,HN
NMR experiment can be modified to a RD 2D H,C,(C-TOCSY-CO,N),HN NMR
experiment, which involves applying radiofrequency pulses so that
the chemical shift evolution of .sup.15N.sub.i does not occur.
Then, the NMR signals are processed to generate a two dimensional
(2D) NMR spectrum with peak pairs where (1) the chemical shift
value of .sup.1H.sup.N.sub.i is measured in a frequency domain
dimension, .omega..sub.2(.sup.1H.sup.N), and (2) the chemical shift
values of .sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.ali), by the frequency differences
between the two peaks forming the peak pairs and the frequencies at
the center of the two peaks, respectively.
[0063] In an alternate embodiment, the RD 3D H,C,(C-TOCSY-CO),N,HN
NMR experiment can be modified to a RD 4D H,C,(C-TOCSY),CO,N,HN NMR
experiment which involves applying radiofrequency pulses so that
the chemical shift evolution of a polypeptide backbone carbonyl
carbon of amino acid residue i-1, .sup.13C'.sub.i-1, occurs under
conditions effective to generate NMR signals encoding the chemical
shift value of .sup.13C'.sub.i-1 in a phase sensitive manner in an
indirect time domain dimension, t.sub.4(.sup.13C'). Then, the NMR
signals are processed to generate a four dimensional (4D) NMR
spectrum with variant peak pairs where (1) the chemical shift
values of .sup.15N.sub.i, .sup.1H.sup.N.sub.i and .sup.13C'.sub.i-1
are measured in three frequency domain dimensions,
.omega..sub.2(.sup.15N), .omega..sub.3(.sup.1H.sup.N), and
.omega..sub.4(.sup.13C'), respectively, and (2) the chemical shift
values of .sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.ali), by the frequency differences
between the two peaks forming the variant peak pairs and the
frequencies at the center of the two peaks, respectively.
The 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA
Experiment
[0064] Another aspect of the present invention relates to a method
of conducting a reduced dimensionality three-dimensional (3D)
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA nuclear magnetic
resonance (NMR) experiment by measuring the chemical shift values
for the following nuclei of a protein molecule having an amino acid
residue, i: (1) a .beta.-proton of amino acid residue i,
.sup.1H.sup..beta..sub.i; (2) a .beta.-carbon of amino acid residue
i, .sup.13C.sup..beta..sub.i; (3) an .alpha.-proton of amino acid
residue i, .sup.1H.sup..alpha..sub.i; (4) an .alpha.-carbon of
amino acid residue i, .sup.13C'.sub.i; and (5) a polypeptide
backbone carbonyl carbon of amino acid residue i, .sup.13C'.sub.i,
where the chemical shift values of
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i/.sup.13C.sup..beta..sub.i which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup..alpha..sub.i,
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are connected to the chemical shift
evolution of .sup.13C'.sub.i, under conditions effective (1) to
generate NMR signals encoding the chemical shift values of
.sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.13C'.sub.i in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup..alpha./.beta.) and
t.sub.2(.sup.13C'), respectively, and the chemical shift value of
.sup.1H.sup..alpha..sub.i in a direct time domain dimension,
t.sub.3(.sup.1H.sup..alpha.), and (2) to sine modulate the chemical
shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i, respectively. Then, the NMR signals are
processed to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift values of .sup.13C'.sub.i and
.sup.1H.sup..alpha..sub.i are measured in two frequency domain
dimensions, .omega..sub.2(.sup.13C') and
.omega..sub.3(.sup.1H.sup..alpha.), respectively, and (2) (i) the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequency differences between each of the two peaks forming each of
the anti-phase peak pairs, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming the anti-phase peak pairs,
where the sine-modulated 3D NMR spectrum enables detection of the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i in a phase sensitive manner.
[0065] In addition, the method of conducting a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment can
involve applying radiofrequency pulses under conditions effective
(1) to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.13C'.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.13C'),
respectively, and the chemical shift value of
.sup.1H.sup..alpha..sub.i in t.sub.3(.sup.1H.sup..alpha.) and (2)
to cosine modulate the .sup.13C.sup..alpha..sub.i and .sup.13
C.sup..beta..sub.i chemical shift evolutions in
t.sub.1(.sup.13C.sup..alpha./.beta.) with the chemical shift
evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals. Then, the
NMR signals and the additional NMR signals are processed to further
generate a cosine-modulated 3D NMR spectrum with in-phase peak
pairs derived from the cosine modulating, a sum 3D NMR spectrum
generated by adding the sine-modulated 3D NMR spectrum and the
cosine-modulated 3D NMR spectrum and a difference 3D NMR spectrum
generated by subtracting the cosine-modulated 3D NMR spectrum from
the sine-modulated 3D NMR spectrum. The combined use of the sum 3D
NMR spectrum and the difference 3D NMR spectrum enables placement
of the two peaks forming the peak pairs into separate spectra,
thereby allowing phase-sensitive editing of the two peaks forming
the peak pairs.
[0066] In addition, the method of conducting a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment can
involve applying radiofrequency pulses under conditions effective
(1) to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.15N.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N) and the
chemical shift value of .sup.1H.sup..alpha..sub.i in
t.sub.3(.sup.1H.sup..alpha.), and (2) to avoid cosine modulating
the chemical shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals. Then, the
NMR signals and the additional NMR signals are processed to
generate a 3D NMR spectrum with additional peaks located centrally
between two peaks forming the peak pairs which measure the chemical
shift values of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i along
.omega..sub.1(.sup.13C.sup..alpha./.beta.). Those additional peaks
can be derived from .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i nuclear spin polarization. One specific
embodiment (3D H.sup..alpha./.beta.C.sup..alpha./.beta.COHA) of
this method is illustrated in FIG. 2E, where the applying
radiofrequency pulses effects a nuclear spin polarization transfer,
where a radiofrequency pulse is used to create transverse
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i
magnetization, and .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i polarization is transferred to
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i, to
.sup.13C'.sub.i, and back to .sup.1H.sup..alpha..sub.i, where the
NMR signal is detected. Another specific embodiment of this method
involves applying radiofrequency pulses by (1) applying a first set
of radiofrequency pulses according to the scheme shown in FIG. 3E
to generate a first NMR signal, and (2) applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3E,
where phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal. Then, prior to the
processing, the first NMR signal and the second NMR signal are
added and subtracted, whereby the NMR signals are processed to
generate a first NMR subspectrum derived from the subtracting which
contains the peak pairs, and a second NMR subspectrum derived from
the adding which contains the additional peaks located centrally
between the two peaks forming the peak pairs.
[0067] In an alternate embodiment, the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment can
be modified to a RD 2D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,(CO),HA NMR experiment,
which involves applying radiofrequency pulses so that the chemical
shift evolution of .sup.13C'.sub.i does not occur. Then, the NMR
signals are processed to generate a two dimensional (2D) NMR
spectrum with peak pairs where (1) the chemical shift value of
.sup.1H.sup..alpha..sub.i is measured in a frequency domain
dimension, .omega..sub.2(.sup.1H.sup..alpha.), and (2) (i) the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequency differences between two peaks forming the peak pairs,
respectively, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming the peak pairs.
The 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN
Experiment
[0068] A further aspect of the present invention relates to a
method of conducting a reduced dimensionality three-dimensional
(3D) H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN nuclear
magnetic resonance (NMR) experiment by measuring the chemical shift
values for the following nuclei of a protein molecule having an
amino acid residue, i: (1) a .beta.-proton of amino acid residue i,
.sup.1H.sup..beta..sub.i; (2) a .beta.-carbon of amino acid residue
i, .sup.13C.sup..beta..sub.i; (3) an .alpha.-proton of amino acid
residue i, .sup.1H.sup..alpha..sub.i; (4) an .alpha.-carbon of
amino acid residue i, .sup.13C.sup..alpha..sub.i; (5) a polypeptide
backbone amide nitrogen of amino acid residue i, .sup.15N.sub.i;
and (6) a polypeptide backbone amide proton of amino acid residue
i, .sup.1H.sup.N.sub.i, where the chemical shift values of
.sup.1H.sup..alpha..sub.i/.sup.13C.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i/.sup.13C.sup..beta..sub.i, which are
encoded in peak pairs of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup..alpha..sub.i,
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i are connected to the chemical shift
evolutions of .sup.15N.sub.i and .sup.1H.sup.N.sub.i, under
conditions effective (1) to generate NMR signals encoding the
chemical shift values of .sup.13C.sup..alpha..sub.i,
.sup.13C.sup..beta..sub.i and .sup.15N.sub.i, in a phase sensitive
manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i,
in a direct time domain dimension, t.sub.3(.sup.1H.sup.N), and (2)
to sine modulate the chemical shift evolutions of
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i in
t.sub.1(.sup.13C.sup..alpha./.beta.) with the chemical shift
evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i, respectively. Then, the NMR signals are
processed to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift values of .sup.15N.sub.i and .sup.1H.sup.N.sub.i
are measured in two frequency domain dimensions,
.omega..sub.2(.sup.15N) and .omega..sub.3(.sup.1H.sup.N),
respectively, and (2) (i) the chemical shift values of
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i are measured
in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequency
differences between each of the two peaks forming each of the
anti-phase peak pairs, and (ii) the chemical shift values of
.sup.13C.sup..alpha..sub.i, and .sup.13C.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequencies at
the center of the two peaks forming the anti-phase peak pairs,
where the sine-modulated 3D NMR spectrum enables detection of the
chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i in a phase sensitive manner.
[0069] In addition, the method of conducting a RD 3D
H.sup..alpha./.beta., C.sup..alpha./.beta.,N,HN NMR experiment can
involve applying radiofrequency pulses under conditions effective
(1) to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i and
.sup.15N.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N),
respectively, and the chemical shift value of .sup.1H.sup.N.sub.i
in t.sub.3(.sup.1H.sup.N) and (2) to cosine modulate the
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i chemical
shift evolutions in t.sub.1(.sup.13C.sup..alpha./.beta.) with the
chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals. Then, the
NMR signals and the additional NMR signals are processed to further
generate a cosine-modulated 3D NMR spectrum with in-phase peak
pairs derived from the cosine modulating, a sum 3D NMR spectrum
generated by adding the sine-modulated 3D NMR spectrum and the
cosine-modulated 3D NMR spectrum and a difference 3D NMR spectrum
generated by subtracting the cosine-modulated 3D NMR spectrum from
the sine-modulated 3D NMR spectrum. The combined use of the sum 3D
NMR spectrum and the difference 3D NMR spectrum enables placement
of the two peaks forming the peak pairs into separate spectra,
thereby allowing phase-sensitive editing of the two peaks forming
the peak pairs.
[0070] In addition, the method of conducting a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment can
involve applying radiofrequency pulses under conditions effective
(1) to generate additional NMR signals encoding the chemical shift
values of .sup.13C.sup..alpha..sub.i, .sup.13C.sup..beta..sub.i,
and .sup.15N.sub.i in a phase sensitive manner in
t.sub.1(.sup.13C.sup..alpha./.beta.) and t.sub.2(.sup.15N) and the
chemical shift value of .sup.1H.sup.N.sub.i in
t.sub.3(.sup.1H.sup.N), and (2) to avoid cosine modulating the
chemical shift evolutions of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i, in t.sub.1(.sup.13C.sup..alpha./.beta.)
with the chemical shift evolutions of .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i for the additional NMR signals. Then, the
NMR signals and the additional NMR signals are processed to
generate a 3D NMR spectrum with additional peaks located centrally
between two peaks forming the peak pairs which measure the chemical
shift values of .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta..sub.i along
.omega..sub.1(.sup.13C.sup..alpha./.beta.). Those additional peaks
can be derived from .sup.13C.sup..alpha..sub.i and
.sup.13C.sup..beta. nuclear spin polarization. One specific
embodiment (3D H.sup..alpha./.beta.C.sup..alpha./.beta.NHN) of this
method is illustrated in FIG. 2F, where the applying radiofrequency
pulses effects a nuclear spin polarization transfer where a
radiofrequency pulse is used to create transverse
.sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i
magnetization, and .sup.1H.sup..alpha..sub.i and
.sup.1H.sup..beta..sub.i magnetization is transferred to
.sup.13C.sup..alpha..sub.i and .sup.13C.sup..beta..sub.i, to
.sup.15N.sub.i, and to .sup.1H.sup.N.sub.i, where the NMR signal is
detected. Another specific embodiment of this method involves
applying radiofrequency pulses by (1) applying a first set of
radiofrequency pulses according to the scheme shown in FIG. 3F to
generate a first NMR signal, and (2) applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3F,
where phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal. Then, prior to the
processing, the first NMR signal and the second NMR signal are
added and subtracted, whereby the NMR signals are processed to
generate a first NMR subspectrum derived from the subtracting which
contains the peak pairs, and a second NMR subspectrum derived from
the adding which contains the additional peaks located centrally
between the two peaks forming the peak pairs.
[0071] In an alternate embodiment, the RD 3D H.sup..alpha./.beta.,
C.sup..alpha./.beta.,N,HN NMR experiment can be modified to a RD 2D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,(N),HN NMR experiment
which involves applying radiofrequency pulses so that the chemical
shift evolution of .sup.15N.sub.i does not occur. Then, the NMR
signals are processed to generate a two dimensional (2D) NMR
spectrum with peak pairs where (1) the chemical shift value of
.sup.1H.sup.N.sub.i is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.N), and (2) (i) the chemical shift values
of .sup.1H.sup..alpha..sub.i and .sup.1H.sup..beta..sub.i are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..alpha./.beta.), by the frequency
differences between the two peaks forming the peak pairs, and (ii)
the chemical shift values of .sup.13C.sup..alpha..sub.i, and
.sup.13C.sup..beta..sub.i are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup..alpha./.beta.), by the
frequencies at the center of the two peaks forming the peak
pairs.
The 3D H,C,H-COSY Experiment
[0072] The present invention also relates to a method of conducting
a reduced dimensionality three-dimensional (3D) H,C,C,H-COSY
nuclear magnetic resonance (NMR) experiment by measuring the
chemical shift values for .sup.1H.sup.m, .sup.13C.sup.m,
.sup.1H.sup.n, and .sup.13C.sup.n of a protein molecule where m and
n indicate atom numbers of two CH, CH.sub.2 or CH.sub.3 groups that
are linked by a single covalent carbon-carbon bond in an amino acid
residue, where the chemical shift values of .sup.1H.sup.m and
.sup.13C.sup.m which are encoded in a peak pair of a 3D NMR
spectrum are detected in a phase sensitive manner. The method
involves providing a protein sample and applying radiofrequency
pulses to the protein sample which effects a nuclear spin
polarization transfer where the chemical shift evolutions of
.sup.1H.sup.m and .sup.13C.sup.m are connected to the chemical
shift evolutions of .sup.1H.sup.n and .sup.13C.sup.n, under
conditions effective (1) to generate NMR signals encoding the
chemical shift values of .sup.13C.sup.m and .sup.13C.sup.n in a
phase sensitive manner in two indirect time domain dimensions,
t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n), respectively,
and the chemical shift value of .sup.1H.sup.n in a direct time
domain dimension, t.sub.3(.sup.1H.sup.n), and (2) to sine modulate
the chemical shift evolution of .sup.13C.sup.m in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sub.m. Then, the NMR signals are processed to generate a
sine-modulated 3D NMR spectrum with anti-phase peak pairs derived
from the sine modulating where (1) the chemical shift values of
.sup.13C.sup.n and .sup.1H.sup.n are measured in two frequency
domain dimensions, .omega..sub.2(.sup.13C.sup.n) and
.omega..sub.3(.sup.1H.sup.n), respectively, and (2) the chemical
shift values of .sup.1H.sup.m and .sup.13C.sup.m are measured in a
frequency domain dimension, .omega..sub.1(.sup.13C.sup.m), by the
frequency differences between each of the two peaks forming each of
the anti-phase peak pairs and the frequencies at the center of the
two peaks, respectively, where the sine-modulated 3D NMR spectrum
enables detection of the chemical shift value of .sup.1H.sub.m in a
phase sensitive manner.
[0073] In addition, the method of conducting a RD 3D H,C,C,H-COSY
NMR experiment can involve applying radiofrequency pulses under
conditions effective (1) to generate additional NMR signals
encoding the chemical shift values of .sup.13C.sup.m and
.sup.13C.sup.n in a phase sensitive manner in
t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n), respectively,
and the chemical shift value of .sup.1H.sup.n in
t.sub.3(.sup.1H.sup.n), and (2) to cosine modulate the
.sup.13C.sup.m chemical shift evolution in t.sub.1(.sup.13C.sup.m)
with the chemical shift evolution of .sup.1H.sub.m for the
additional NMR signals. Then, the NMR signals and the additional
NMR signals are processed to further generate a cosine-modulated 3D
NMR spectrum with in-phase peak pairs derived from the cosine
modulating, a sum 3D NMR spectrum generated by adding the
sine-modulated 3D NMR spectrum and the cosine-modulated 3D NMR
spectrum and a difference 3D NMR spectrum generated by subtracting
the cosine-modulated 3D NMR spectrum from the sine-modulated 3D NMR
spectrum. The combined use of the sum 3D NMR spectrum and the
difference 3D NMR spectrum enables placement of the two peaks
forming the peak pairs into separate spectra, thereby allowing
phase-sensitive editing of the two peaks forming the peak
pairs.
[0074] In addition, the method of conducting a RD 3D H,C,C,H-COSY
NMR experiment can involve applying radiofrequency pulses under
conditions effective (1) to generate additional NMR signals
encoding the chemical shift values of .sup.13C.sup.m and
.sup.13C.sup.n in a phase sensitive manner in
t.sub.1(.sup.13C.sup.m) and t.sub.2(.sup.13C.sup.n) and the
chemical shift value of .sup.1H.sup.n in t.sub.3(.sup.1H), and (2)
to avoid cosine modulating the chemical shift evolution of
.sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the chemical shift
evolution of .sup.1H.sup.m for the additional NMR signals. Then,
the NMR signals and the additional NMR signals are processed to
generate a 3D NMR spectrum with additional peaks located centrally
between two peaks forming the peak pairs which measure the chemical
shift value of .sup.13C.sup.m along .omega..sub.1(.sup.13C.sup.m).
Those additional peaks can be derived from .sup.13C.sup.m nuclear
spin polarization. One specific embodiment (3D HCCH-COSY) of this
method is illustrated in FIG. 2H, where the applying radiofrequency
pulses effects a nuclear spin polarization transfer, where a
radiofrequency pulse is used to create transverse .sup.1H.sup.m
magnetization, and .sup.1H.sup.m magnetization is transferred to
.sup.13C.sup.m, to .sup.13C.sub.n, and to .sup.1H.sup.n, where the
NMR signal is detected. Another specific embodiment of this method
involves applying radiofrequency pulses by (1) applying a first set
of radiofrequency pulses according to the scheme shown in FIG. 3H
to generate a first NMR signal, and (2) applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3H,
where phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal. Then, prior to the
processing, the first NMR signal and the second NMR signal are
added and subtracted, whereby the NMR signals are processed to
generate a first NMR subspectrum derived from the subtracting which
contains the peak pairs, and a second NMR subspectrum derived from
the adding which contains the additional peaks located centrally
between the two peaks forming the peak pairs.
[0075] In an alternate embodiment, the RD 3D H,C,C,H-COSY NMR
experiment can be modified to a RD 2D H,C,(C),H-COSY NMR experiment
which involves applying radiofrequency pulses so that the chemical
shift evolution of .sup.13C.sup.n does not occur. Then, the NMR
signals are processed to generate a two dimensional (2D) NMR
spectrum with peak pairs where (1) the chemical shift value of
.sup.1H.sup.n is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.n), and (2) the chemical shift values of
.sup.1H.sup.m and .sup.13C.sup.m are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.m), by the frequency
differences between the two peaks forming the peak pairs and the
frequencies at the center of the two peaks, respectively.
The 3D H,C,C,H-TOCSY Experiment
[0076] Another aspect of the present invention relates to a method
of conducting a reduced dimensionality three-dimensional (3D)
H,C,C,H-TOCSY nuclear magnetic resonance (NMR) experiment by
measuring the chemical shift values for .sup.1H.sup.m,
.sup.13C.sup.m, .sup.1H.sup.n, and .sup.13C.sup.n of a protein
molecule where m and n indicate atom numbers of two CH, CH.sub.2 or
CH.sub.3 groups that may or may not be directly linked by a single
covalent carbon-carbon bond in an amino acid residue, where the
chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m which are
encoded in a peak pair of a 3D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup.m and .sup.13C.sup.m are connected
to the chemical shift evolutions of .sup.1H.sup.n and
.sup.13C.sup.n, under conditions effective (1) to generate NMR
signals encoding the chemical shift values of .sup.13C.sup.m and
.sup.13C.sup.n in a phase sensitive manner in two indirect time
domain dimensions, t.sub.1(.sup.13C.sup.m) and
t.sub.2(.sup.13C.sup.n), and the chemical shift value of
.sup.1H.sup.n in a direct time domain dimension,
t.sub.3(.sup.1H.sup.n), and (2) to sine modulate the chemical shift
evolution of .sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the
chemical shift evolution of .sup.1H.sup.m. Then, the NMR signals
are processed to generate a sine-modulated 3D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift values of .sup.13C.sup.n and .sup.1H.sup.n are
measured in two frequency domain dimensions,
.omega..sub.2(.sup.13C.sup.n) and .omega..sub.3(.sup.1H.sup.n),
respectively, and (2) the chemical shift values of .sup.1H.sup.m
and .sup.13C.sup.m are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.m), by the frequency differences between
each of the two peaks forming each of the anti-phase peak pairs and
the frequencies at the center of the two peaks, respectively, where
the sine-modulated 3D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sub.m in a phase sensitive
manner.
[0077] In addition, the method of conducting a RD 3D H,C,C,H-TOCSY
NMR can involve applying radiofrequency pulses under conditions
effective (1) to generate additional NMR signals encoding the
chemical shift values of .sup.13C.sup.m and .sup.13C.sup.n in a
phase sensitive manner in t.sub.1(.sup.13C.sup.m) and
t.sub.2(.sup.13C.sup.n), respectively, and the chemical shift value
of .sup.1H.sup.n in t.sub.3(.sup.1H.sup.n) and (2) to cosine
modulate the .sup.13C.sup.m chemical shift evolution in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sub.m for the additional NMR signals. Then, the NMR signals
and the additional NMR signals are processed to further generate a
cosine-modulated 3D NMR spectrum with in-phase peak pairs derived
from the cosine modulating, a sum 3D NMR spectrum generated by
adding the sine-modulated 3D NMR spectrum and the cosine-modulated
3D NMR spectrum, and a difference 3D NMR spectrum generated by
subtracting the cosine-modulated 3D NMR spectrum from the
sine-modulated 3D NMR spectrum. The combined use of the sum 3D NMR
spectrum and the difference 3D NMR spectrum enables placement of
the two peaks forming the peak pairs into separate spectra, thereby
allowing phase-sensitive editing of the two peaks forming the peak
pairs.
[0078] In addition, the method of conducting a RD 3D H,C,C,H-TOCSY
NMR can involve applying radiofrequency pulses under conditions
effective (1) to generate additional NMR signals encoding the
chemical shift values of .sup.13C.sup.m and .sup.13C.sup.n in a
phase sensitive manner in t.sub.1(.sup.13C.sup.m) and
t.sub.2(.sup.13C.sup.n) and the chemical shift value of
.sup.1H.sup.n in t.sub.3(.sup.1H.sup.n), and (2) to avoid cosine
modulating the chemical shift evolution of .sup.13C.sup.m in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sup.m for the additional NMR signals. Then, the NMR signals
and the additional NMR signals are processed to generate a 3D NMR
spectrum with additional peaks located centrally between two peaks
forming the peak pairs which measure the chemical shift value of
.sup.13C.sup.m along .omega..sub.1(.sup.13C.sup.m). Those
additional peaks can be derived from .sup.13C.sup.m nuclear spin
polarization. One specific embodiment (3D HCCH-TOCSY) of this
method is illustrated in FIG. 2I, where the applying radiofrequency
pulses effects a nuclear spin polarization transfer where a
radiofrequency pulse is used to create transverse .sup.1H.sup.m
magnetization, and .sup.1H.sup.m magnetization is transferred to
.sup.13C.sup.m, to .sup.13C.sup.n, and to .sup.1H.sup.n, where the
NMR signal is detected. Another specific embodiment of this method
involves applying radiofrequency pulses by (1) applying a first set
of radiofrequency pulses according to the scheme shown in FIG. 3I
to generate a first NMR signal, and (2) applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3I,
where phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal. Then, prior to the
processing, the first NMR signal and the second NMR signal are
added and subtracted, whereby the NMR signals are processed to
generate a first NMR subspectrum derived from the subtracting which
contains the peak pairs, and a second NMR subspectrum derived from
the adding which contains the additional peaks located centrally
between the two peaks forming the peak pairs.
[0079] In an alternate embodiment, the RD 3D H,C,C,H-TOCSY NMR
experiment can be modified to a RD 2D H,C,(C),H-TOCSY NMR
experiment which involves applying radiofrequency pulses so that
the chemical shift evolution of .sup.13C.sup.n does not occur.
Then, the NMR signals are processed to generate a two dimensional
(2D) NMR spectrum with peak pairs where (1) the chemical shift
value of .sup.1H.sup.n is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup.n), and (2) the chemical shift values of
.sup.1H.sup.m and .sup.13C.sup.m are measured in a frequency domain
dimension, .omega..sub.1(.sup.13C.sup.m), by the frequency
differences between the two peaks forming the peak pairs and the
frequencies at the center of the two peaks, respectively.
The 2D HB,CB,(CG,CD),HD Experiment
[0080] A further aspect of the present invention relates to a
method of conducting a reduced dimensionality two-dimensional (2D)
HB,CB,(CG,CD),HD nuclear magnetic resonance (NMR) experiment by
measuring the chemical shift values for the following nuclei of a
protein molecule: (1) a .beta.-proton of an amino acid residue with
an aromatic side chain, .sup.1H.sup..beta.; (2) a .beta.-carbon of
an amino acid residue with an aromatic side chain,
.sup.13C.sup..beta.; and (3) a .delta.-proton of an amino acid
residue with an aromatic side chain, .sup.1H.sup..delta., where the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
which are encoded in a peak pair of a 2D NMR spectrum are detected
in a phase sensitive manner. The method involves providing a
protein sample and applying radiofrequency pulses to the protein
sample which effect a nuclear spin polarization transfer where the
chemical shift evolutions of .sup.1H.sup..beta. and
.sup.13C.sup..beta. are connected to the chemical shift evolution
of .sup.1H.sup..delta., under conditions effective (1) to generate
NMR signals encoding the chemical shift value of
.sup.13C.sup..beta. in a phase sensitive manner in an indirect time
domain dimension, t.sub.1(.sup.13C.sup..beta.), and the chemical
shift value of .sup.1H.sup..delta. in a direct time domain
dimension, t.sub.2(.sup.1H.sup..delta.), and (2) to sine modulate
the chemical shift evolution of .sup.13C.sup..beta. in
t.sub.1(.sup.13C.sup..beta.) with the chemical shift evolution of
.sup.1H.sup..beta.. Then, the NMR signals are processed to generate
a sine-modulated 2D NMR spectrum with an anti-phase peak pair
derived from the sine modulating where (1) the chemical shift value
of .sup.1H.sup..delta. is measured in a frequency domain dimension,
.omega..sub.2(.sup.1H.sup..delta.), and (2) the chemical shift
values of .sup.1H.sup..beta. and .sup.13C.sup..beta. are measured
in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming the anti-phase peak pair and the
frequency at the center of the two peaks, respectively, where the
sine-modulated 2D NMR spectrum enables detection of the chemical
shift value of .sup.1H.sup..beta. in a phase sensitive manner.
[0081] In addition, the method of conducting a RD 2D
HB,CB,(CG,CD),HD NMR experiment can involve applying radiofrequency
pulses under conditions effective (1) to generate additional NMR
signals encoding the chemical shift value of .sup.13C.sup..beta. in
a phase sensitive manner in t.sub.1(.sup.13C.sup..beta.) and the
chemical shift value of .sup.1H.sup..delta. in
t.sub.2(.sup.1H.sup..delta.) and (2) to cosine modulate the
.sup.13C.sup..beta. chemical shift evolution in
t.sub.1(.sup.13C.sup..beta.) with the chemical shift evolution of
.sup.1H.sup..beta. for the additional NMR signals. Then, the NMR
signals and the additional NMR signals are processed to further
generate a cosine-modulated 2D NMR spectrum with an in-phase peak
pair derived from the cosine modulating, a sum 2D NMR spectrum
generated by adding the sine-modulated 2D NMR spectrum and the
cosine-modulated 2D NMR spectrum, and a difference 2D NMR spectrum
generated by subtracting the cosine-modulated 2D NMR spectrum from
the sine-modulated 2D NMR spectrum. The combined use of the sum 2D
NMR spectrum and the difference 2D NMR spectrum enables placement
of the two peaks forming the peak pairs into separate spectra,
thereby allowing phase-sensitive editing of the two peaks forming
the peak pairs.
[0082] In addition, the method of conducting a RD 2D
HB,CB,(CG,CD),HD NMR experiment can involve applying radiofrequency
pulses under conditions effective (1) to generate additional NMR
signals encoding the chemical shift value of .sup.13C.sup..beta. in
a phase sensitive manner in t.sub.1(.sup.13C.sup..beta.) and the
chemical shift value of .sup.1H.sup..delta. in
t.sub.2(.sup.1H.sup..delta.), and (2) to avoid cosine modulating
the chemical shift evolution of .sup.13C.sup..beta. in
t.sub.1(.sup.13C.sup..beta.) with the chemical shift evolution of
.sup.1H.sup..beta. for the additional NMR signals. Then, the NMR
signals and the additional NMR signals are processed to generate a
2D NMR spectrum with an additional peak located centrally between
the two peaks forming the peak pair which measure the chemical
shift value of .sup.13C.sup..beta. along .omega..sub.1(.sup.13C).
That additional peak can be derived from .sup.13C.sup..beta.
nuclear spin polarization. One specific embodiment (2D
HBCB(CGCD)HD) of this method is illustrated in FIG. 2J, where the
applying radiofrequency pulses effects a nuclear spin polarization
transfer where a radiofrequency pulse is used to create transverse
.sup.1H.sup..beta. magnetization, and .sup.1H.sup..beta.
magnetization is transferred to .sup.13C.sup..beta., to
.sup.13C.sup..delta., and to .sup.1H.sup..delta., where the NMR
signal is detected. Another specific embodiment of this method
involves applying radiofrequency pulses by (1) applying a first set
of radiofrequency pulses according to the scheme shown in FIG. 3J
to generate a first NMR signal, and (2) applying a second set of
radiofrequency pulses according to the scheme shown in FIG. 3J,
where phase .phi..sub.1 of the first .sup.1H pulse is altered by
180.degree. to generate a second NMR signal. Then, prior to the
processing, the first NMR signal and the second NMR signal are
added and subtracted, whereby the NMR signals are processed to
generate a first NMR subspectrum derived from the subtracting which
contains the peak pair, and a second NMR subspectrum derived from
the adding which contains the additional peak located centrally
between the two peaks forming the peak pair.
[0083] In an alternate embodiment, the RD 2D HB,CB,(CG,CD),HD NMR
experiment can be modified to a RD 3D HB,CB,(CG),CD,HD NMR
experiment which involves applying radiofrequency pulses so that
the chemical shift evolution of a 3-carbon of an amino acid residue
with an aromatic side chain, .sup.13C.sup..delta. occurs under
conditions effective to generate NMR signals encoding the chemical
shift value of .sup.13C.sup..delta. in a phase sensitive manner in
an indirect time domain dimension, t.sub.3(.sup.13C.sup..delta.).
Then, the NMR signals are processed to generate a three dimensional
(3D) NMR spectrum with a peak pair where (1) the chemical shift
values of .sup.1H.sup..delta. and .sup.13C.sup..delta. are measured
in two frequency domain dimensions,
.omega..sub.2(.sup.1H.sup..delta.) and
.omega..sub.3(.sup.13C.sup..delta.), respectively, and (2) the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming the peak pair and the frequency at
the center of the two peaks, respectively.
[0084] In an alternate embodiment, the RD 2D HB,CB,(CG,CD),HD NMR
experiment can be modified to a RD 3D HB,CB,CG,(CD),HD NMR
experiment which involves applying radiofrequency pulses so that
the chemical shift evolution of a .gamma.-carbon of an amino acid
residue with an aromatic side chain, .sup.13C.sup..gamma. occurs
under conditions effective to generate NMR signals encoding the
chemical shift value of .sup.13C.sup..gamma. in a phase sensitive
manner in an indirect time domain dimension,
t.sub.3(.sup.13C.sup..gamma.), and said processing the NMR signals
generates a three dimensional (3D) NMR spectrum with a peak pair
wherein (1) the chemical shift values of .sup.1H.sup..delta. and
.sup.13C.sup..gamma. are measured in two frequency domain
dimensions, .omega..sub.2(.sup.1H.sup..delta.) and
.omega..sub.3(.sup.13C.sup..gamma.), respectively, and (2) the
chemical shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta.
are measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup..beta.), by the frequency difference
between the two peaks forming said peak pair and the frequency at
the center of the two peaks, respectively.
The 2D H,C,H-COSY Experiment
[0085] The present invention also relates to a method of conducting
a reduced dimensionality two-dimensional (2D) H,C,H-COSY nuclear
magnetic resonance (NMR) experiment by measuring the chemical shift
values for .sup.1H.sup.m, .sup.13C.sup.m, and .sup.1H.sup.n of a
protein molecule where m and n indicate atom numbers of two CH,
CH.sub.2 or CH.sub.3 groups in an amino acid residue, where the
chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m which are
encoded in a peak pair of a 2D NMR spectrum are detected in a phase
sensitive manner. The method involves providing a protein sample
and applying radiofrequency pulses to the protein sample which
effect a nuclear spin polarization transfer where the chemical
shift evolutions of .sup.1H.sup.m and .sup.13C.sup.m are connected
to the chemical shift evolution of .sup.1H.sup.n, under conditions
effective (1) to generate NMR signals encoding the chemical shift
value of .sup.13C.sup.m in a phase sensitive manner in an indirect
time domain dimension, t.sub.1(.sup.13C.sup.m), and the chemical
shift value of .sup.1H.sup.n in a direct time domain dimension,
t.sub.2(.sup.1H.sup.n), and (2) to sine modulate the chemical shift
evolution of .sup.13C.sup.m in t.sub.1(.sup.13C.sup.m) with the
chemical shift evolution of .sup.1H.sup.m. Then, the NMR signals
are processed to generate a sine-modulated 2D NMR spectrum with
anti-phase peak pairs derived from the sine modulating where (1)
the chemical shift value of .sup.1H.sup.m is measured in a
frequency domain dimension, .omega..sub.2(.sup.1H.sup.n), and (2)
the chemical shift values of .sup.1H.sup.m and .sup.13C.sup.m are
measured in a frequency domain dimension,
.omega..sub.1(.sup.13C.sup.m), by the frequency differences between
each of the two peaks forming each of the anti-phase peak pairs and
the frequencies at the center of the two peaks, respectively, where
the sine-modulated 2D NMR spectrum enables detection of the
chemical shift value of .sup.1H.sub.m in a phase sensitive
manner.
[0086] In addition, the method of conducting a RD 2D H,C,H-COSY NMR
experiment can involve applying radiofrequency pulses under
conditions effective (1) to generate additional NMR signals
encoding the chemical shift value of .sup.13C.sup.m in a phase
sensitive manner in t.sub.1(.sup.13C.sup.m) and the chemical shift
value of .sup.1H.sup.n in t.sub.2(.sup.1H.sup.n) and (2) to cosine
modulate the .sup.13C.sup.m chemical shift evolution in
t.sub.1(.sup.13C.sup.m) with the chemical shift evolution of
.sup.1H.sub.m for the additional NMR signals. Then, the NMR signals
and the additional NMR signals are processed to further generate a
cosine-modulated 2D NMR spectrum with in-phase peak pairs derived
from the cosine modulating, a sum 2D NMR spectrum generated by
adding the sine-modulated 2D NMR spectrum and the cosine-modulated
2D NMR spectrum and a difference 2D NMR spectrum generated by
subtracting the cosine-modulated 2D NMR spectrum from the
sine-modulated 2D NMR spectrum. The combined use of the sum 2D NMR
spectrum and the difference 2D NMR spectrum enables placement of
the two peaks forming the peak pairs into separate spectra, thereby
allowing phase-sensitive editing of the two peaks forming the peak
pairs.
[0087] One specific embodiment (2D .sup.1H-TOCSY-HCH-COSY) of this
method is illustrated in FIG. 2K, where the applying radiofrequency
pulses effects a nuclear spin polarization transfer where a
radiofrequency pulse is used to create transverse .sup.1H.sup.m
magnetization, and .sup.1H.sup.m polarization is transferred to
.sup.13C.sup.m, to .sup.1H.sup.m, and to .sup.1H.sup.n, where the
NMR signal is detected. Although the specific embodiment
illustrated in FIG. 2K shows this method applied to an amino acid
residue with an aromatic side chain, this method also applies to
amino acid residues with aliphatic side chains. Another specific
embodiment of this method involves applying radiofrequency pulses
according to the scheme shown in FIG. 3K.
[0088] FIG. 4 outlines which chemical shifts are correlated in the
various NMR experiments described above.
Combinations of RD NMR Experiments
[0089] Accordingly, a suite of multidimensional RD NMR experiments
enables one to devise strategies for RD NMR-based HTP resonance
assignment of proteins.
[0090] Thus, another aspect of the present invention relates to a
method for sequentially assigning chemical shift values of an
.alpha.-proton, .sup.1H.sup..alpha., an .alpha.-carbon,
.sup.13C.sup..alpha., a polypeptide backbone amide nitrogen,
.sup.15N, and a polypeptide backbone amide proton, .sup.1H.sup.N,
of a protein molecule. The method involves providing a protein
sample and conducting a set of reduced dimensionality (RD) nuclear
magnetic resonance (NMR) experiments on the protein sample, where
the chemical shift values of .sup.1H.sup..alpha. and
.sup.13C.sup..alpha. which are encoded in a peak pair of a 3D NMR
spectrum are detected in a phase sensitive manner, including: (1) a
RD 3D HA,CA,(CO),N,HN NMR experiment to measure and connect
chemical shift values of the .alpha.-proton of amino acid residue
i-1, .sup.1H.sup..alpha..sub.i-1, the .alpha.-carbon of amino acid
residue i-1, .sup.13C.sup..alpha..sub.i-1, the polypeptide backbone
amide nitrogen of amino acid residue i, .sup.15N.sub.i, and the
polypeptide backbone amide proton of amino acid residue i,
.sup.1H.sup.N.sub.i and (2) a RD 3D HNNCAHA NMR experiment to
measure and connect the chemical shift values of the .alpha.-proton
of amino acid residue i, .sup.1H.sup..alpha..sub.i, the
.alpha.-carbon of amino acid residue i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i. Then, sequential
assignments of the chemical shift values of .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N are obtained by
(i) matching the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 with
the chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.13C.sup..alpha..sub.i, (ii) using the chemical shift values of
.sup.1H.sup..alpha..sub.i-1 and .sup.13C.sup..alpha..sub.i-1 to
identify the type of amino acid residue i-1 (Wuthrich, NMR of
Proteins and Nucleic Acids, Wiley, New York (1986); Grzesiek et
al., J. Biomol. NMR, 3: 185-204 (1993), which are hereby
incorporated by reference in their entirety), and (iii) mapping
sets of sequentially connected chemical shift values to the amino
acid sequence of the polypeptide chain and using the chemical shift
values to locate secondary structure elements (such as
.alpha.-helices and .beta.-sheets) within the polypeptide chain
(Spera et al., J. Am. Chem. Soc., 113:5490-5492 (1991); Wishart et
al., Biochemistry, 31:1647-1651 (1992), which are hereby
incorporated by reference in their entirety).
[0091] In one embodiment, the protein sample could, in addition to
the RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA NMR
experiment, be further subjected to a RD 3D HNN<CO,CA> NMR
experiment to measure and connect the chemical shift values of a
polypeptide backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift value of .sup.13C'.sub.i-1, are obtained by matching the
chemical shift value of .sup.13C.sup..alpha..sub.i measured by the
RD 3D HNN<CO,CA> NMR experiment with the sequentially
assigned chemical shift values of .sup.13C.sup..alpha., .sup.15N,
and .sup.1H.sup.N measured by the RD 3D HA,CA,(CO),N,HN NMR
experiment and the RD 3D HNNCAHA NMR experiment.
[0092] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA
NMR experiment, be further subjected to (i) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
the .alpha.-proton of amino acid residue i,
.sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid residue
i, .sup.13C.sup..alpha..sub.i, and a polypeptide backbone carbonyl
carbon of amino acid residue i, .sup.13C'.sub.i, and (ii) a RD 3D
HNN<CO,CA> NMR experiment to measure and connect the chemical
shift values of .sup.13C'.sub.i, the .alpha.-carbon of amino acid
residue i+1, .sup.13C.sup..alpha..sub.i+1, the polypeptide backbone
amide nitrogen of amino acid residue i+1, .sup.15N.sub.i+1, and the
polypeptide backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1. Then, sequential assignments are obtained by
matching the chemical shift value of .sup.13C'.sub.i measured by
the RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment.
[0093] In another embodiment, the protein sample could, in addition
to the RD 3D H,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA
NMR experiment, be further subjected to a RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment to measure and connect the
chemical shift values of aliphatic protons (including .alpha.-,
.beta.-, and .gamma.-protons) of amino acid residue i-1,
.sup.1H.sup.ali.sub.i-1, aliphatic carbons (including .alpha.-,
.beta.-, and .gamma.-carbons) of amino acid residue i-1,
.sup.13C.sup.ali.sub.i-1, .sup.15N.sub.i, and .sup.1H.sup.N.sub.i.
Then, sequential assignments of the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 for amino acid
residues i having unique pairs of .sup.15N.sub.i and
.sup.1H.sup.N.sub.i chemical shift values are obtained by matching
the chemical shift values of .sup.1H.sup..alpha. and
.sup.13C.sup..alpha. measured by said RD 3D HNNCAHA NMR experiment
and RD 3D HA,CA,(CO),N,HN NMR experiment with the chemical shift
values of .sup.1H.sup..alpha..sub.i-1 and
.sup.13C.sup..alpha..sub.i-1 measured by said RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment and using the
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 chemical shift
values to identify the type of amino acid residue i-1.
[0094] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA
NMR experiment, be further subjected to a RD 3D H,C,C,H-COSY NMR
experiment or a RD 3D H,C,C,H-TOCSY NMR experiment to measure and
connect the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i of amino acid residue i. Then, sequential
assignments of the chemical shift values of .sup.1H.sup.ali.sub.i
and .sup.13C.sup.ali.sub.i, the chemical shift values of a
.gamma.-proton, .sup.1H.sup..gamma., and a .gamma.-carbon,
.sup.13C.sup..gamma..sub.i, in particular, are obtained by (i)
matching the chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.13C.sup..alpha..sub.i measured using the RD 3D H,C,C,H-COSY
NMR experiment or the RD 3D H,C,C,H-TOCSY RD NMR experiment with
the chemical shift values of .sup.1H.sup..alpha..sub.i and
.sup.13C.sup..alpha. measured by the RD 3D HA,CA,(CO),N,HN NMR
experiment, the RD 3D HNNCAHA NMR experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
(ii) using the chemical shift values of .sup.1H.sup.ali and
.sup.13C.sup.ali, including the chemical shift values of
.sup.1H.sup..gamma..sub.i, and .sup.13C.sup..gamma..sub.i, to
identify the type of amino acid residue i.
[0095] In yet another embodiment, this method involves, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment and the RD 3D HNNCAHA
NMR experiment, further subjecting the protein sample to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i-1, .sup.1H.sup..beta..sub.i-1, the
.beta.-carbon of amino acid residue i-1,
.sup.13C.sup..beta..sub.i-1, .sup.1H.sup..alpha..sub.i-1,
.sup.13C.sup..alpha..sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta. are
obtained by using the chemical shift values of
.sup.1H.sup..beta..sub.i-1 and .sup.13C.sup..beta..sub.i-1 to
identify the type of amino acid residue i-1.
[0096] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR
experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, be
further subjected to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta., CO,HA NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i. Then, sequential assignments of the chemical shift
value of .sup.13C'.sub.i are obtained by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by the RD 3D
H.sup..alpha./.beta., C.sup..alpha./.beta.,CO,HA NMR experiment
with the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by the
RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR
experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
[0097] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR
experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, be
further subjected to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i. Then, sequential
assignments are obtained by matching the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i with the
chemical shift values of .sup.1H.sup..beta..sub.i-1,
.sup.13C.sup..beta..sub.i-1, .sup.1H.sup..alpha..sub.i-1, and
.sup.13C.sup..alpha..sub.i-1 measured by the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
[0098] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR
experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, be
further subjected to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift value of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments are obtained by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measured by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
[0099] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR
experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, be
further subjected to a RD 2D HB,CB,(CG,CD),HD NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1, and a
.delta.-proton of amino acid residue I-1 with an aromatic side
chain, .sup.1H.sup..delta..sub.i-1. Then, sequential assignments
are obtained by matching (i) the chemical shift values of
.sup.1H.sup..beta..sub.i-1 and .sup.13C.sup..beta..sub.i-1 measured
by said RD 2D HB,CB,(CG,CD),HD NMR experiment with the chemical
shift values of .sup.1H.sup..beta. and .sup.13C.sup..beta. measured
by the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, (ii) using the chemical shift values to identify amino
acid residue i as having an aromatic side chain, and (iii) mapping
sets of sequentially connected chemical shift values to the amino
acid sequence of the polypeptide chain and locating amino acid
residues with aromatic side chains along the polypeptide chain.
[0100] In another embodiment, the protein sample could, in addition
to the RD 3D HA,CA,(CO),N,HN NMR experiment, the RD 3D HNNCAHA NMR
experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, be
further subjected to a RD 3D H,C,C,H-COSY NMR experiment or a RD 3D
H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons (including .alpha.-, .beta.-, and
.gamma.-protons) of amino acid residue i, .sup.1H.sup.ali.sub.i,
and aliphatic carbons (including .alpha.-, .beta.-, and
.gamma.-carbons) of amino acid residue i, .sup.13C.sup.ali.sub.i,
of amino acid residue i. Then, sequential assignments of the
chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i, the chemical shift values of a
.gamma.-proton, .sup.1H.sup..gamma., and a .gamma.-carbon,
.sup.13C.sup..gamma., in particular, are obtained by (i) matching
the chemical shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i, measured using the RD 3D H,C,C,H-COSY
NMR experiment or the RD 3D H,C,C,H-TOCSY RD NMR experiment with
the chemical shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by the RD 3D HA,CA,(CO),N,HN
NMR experiment, the RD 3D HNNCAHA NMR experiment, and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
(ii) using the chemical shift values of .sup.1H.sup.ali and
.sup.13C.sup.ali, including the chemical shift values of
.sup.1H.sup..gamma. and .sup.13C.sup..gamma., to identify the type
of amino acid residue i.
[0101] Yet another aspect of the present invention relates to a
method for sequentially assigning chemical shift values of a
.beta.-proton, .sup.1H.sup..beta., a .beta.-carbon,
.sup.13C.sup..beta., an .alpha.-proton, .sup.1H.sup..alpha., an
.alpha.-carbon, .sup.13C.sup..alpha., a polypeptide backbone amide
nitrogen, .sup.15N, and a polypeptide backbone amide proton,
.sup.1H.sup.N.sub.i, of a protein molecule. The method involves
providing a protein sample and conducting a set of reduced
dimensionality (RD) nuclear magnetic resonance (NMR) experiments on
the protein sample, where the chemical shift values of
.sup.1H.sup..alpha./.beta. and .sup.13C.sup..alpha./.beta. which
are encoded in peak pairs of a 3D NMR spectrum are detected in a
phase sensitive manner, including: (1) a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i-1, .sup.1H.sup..beta..sub.i-1, the
.beta.-carbon of amino acid residue i-1,
.sup.13C.sup..beta..sub.i-1, the .alpha.-proton of amino acid
residue i-1, .sup.1H.sup..alpha..sub.i-1, the .alpha.-carbon of
amino acid residue i-1, .sup.13C.sup..alpha..sub.i-1, the
polypeptide backbone amide nitrogen of amino acid residue i,
.sup.15N.sub.i, and the polypeptide backbone amide proton of amino
acid residue i, .sup.1H.sup.N.sub.i and (2) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of the .beta.-proton
of amino acid residue i, .sup.1H.sup..beta..sub.i, the
.beta.-carbon of amino acid residue i, .sup.13C.sup..beta..sub.i,
the .alpha.-proton of amino acid residue i,
.sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid residue
i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup..beta., .sup.13C.sup..beta.,
.sup.1H.sup..alpha., .sup.13C.sup..alpha., .sup.15N, and
.sup.1H.sup.N are obtained by (i) matching the chemical shift
values of the .alpha.- and .beta.-protons of amino acid residue
i-1, .sup.1H.sup..alpha./.beta..sub.i-1, and the chemical shift
values of the .alpha.- and .beta.-carbons of amino acid residue
i-1, .sup.13C.sup..alpha./.beta..sub.i-1, with
.sup.1H.sup..alpha./.beta..sub.i and
.sup.13C.sup..alpha./.beta..sub.i, (ii) using
.sup.1H.sup..alpha./.beta..sub.i-1 and
.sup.13C.sup..alpha./.beta..sub.i-1 to identify the type of amino
acid residue i-1 (Wuthrich, NMR of Proteins and Nucleic Acids,
Wiley, New York (1986); Grzesiek et al., J. Biomol. NMR, 3: 185-204
(1993), which are hereby incorporated by reference in their
entirety), (iii) mapping sets of sequentially connected chemical
shift values to the amino acid sequence of the polypeptide chain
and using the chemical shift values to locate secondary structure
elements within the polypeptide chain (Spera et al., J. Am. Chem.
Soc., 113:5490-5492 (1991); Wishart et al., Biochemistry,
31:1647-1651 (1992), which are hereby incorporated by reference in
their entirety).
[0102] In one embodiment, the protein sample could, in addition to
the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D HA,CA,(CO),N,HN NMR experiment (i) to
measure and connect chemical shift values of
.sup.1H.sup..alpha..sub.i-1, .sup.13C.sup..alpha..sub.i-1,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i and (ii) to distinguish
between NMR signals for .sup.1H.sup..beta./.sup.13C.sup..alpha. and
.sup.1H.sup..beta./.sup.13C.sup..beta. measured in the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
the RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta., N,HN NMR
experiment.
[0103] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i. Then, sequential assignments of the chemical shift
value of .sup.13C'.sub.i are obtained by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by the
RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment.
[0104] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of a polypeptide
backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift value of .sup.13C'.sub.i-1 are obtained by matching the
chemical shift value of .sup.13C.sup..alpha..sub.i measured by the
RD 3D HNN<CO,CA> NMR experiment with the sequentially
assigned chemical shift values of .sup.13C.sup..alpha., .sup.15N,
and .sup.1H.sup.N measured by the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D HP,C,N,HN NMR experiment.
[0105] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to (i) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..alpha./.beta.,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i and (ii) a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of .sup.13C'.sub.i,
the .alpha.-carbon of amino acid residue i+1,
.sup.13C.sup..alpha..sub.i+1, the polypeptide backbone amide
nitrogen of amino acid residue i+1, .sup.15N.sub.i+1, and the
polypeptide backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1. Then, sequential assignments are obtained by
matching the chemical shift value of .sup.13C'.sub.i measured by
said RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment.
[0106] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment
to measure and connect the chemical shift values of
.sup.1H.sup.ali.sub.i-1, .sup.13C.sup.ali.sub.i-1, .sup.15N.sub.i,
and .sup.1H.sup.N.sub.i. Then, sequential assignments of the
chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.i-1 for amino acid residues i having unique
pairs of .sup.15N.sub.i and .sup.1H.sup.N.sub.i chemical shift
values are obtained by matching the chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha., and
.sup.13C.sup..alpha. measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.(CO)NHN NMR experiment and
RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.N,HN NMR experiment
with the chemical shift values of
.sup.1H.sup..beta..sub.i-1,.sup.13C.sup..beta..sub.i-1,
.sup.1H.sup..alpha..sub.i-1, and .sup.13C.sup..alpha..sub.i-1
measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and
using the .sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1
chemical shift values to identify the type of amino acid residue
i-1.
[0107] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift value of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments are obtained by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measured by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i and .sup.13C.sup..alpha..sub.i-1 measured
by the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment.
[0108] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 2D HB,CB,(CG,CD),HD NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i, and a
.delta.-proton of amino acid residue i with an aromatic side chain,
.sup.1H.sup..delta..sub.i. Then, sequential assignments are
obtained by (i) matching the chemical shift values of
.sup.1H.sup..beta..sub.i and .sup.13C.sup..beta..sub.i measured by
said RD 2D HB,CB,(CG,CD),HD NMR experiment with the chemical shift
values of .sup.1H.sup..beta. and .sup.13C.sup..beta. measured by
the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, (ii)
using the chemical shift values to identify amino acid residue i as
having an aromatic side chain, and (iii) mapping sets of
sequentially connected chemical shift values to the amino acid
sequence of the polypeptide chain and locating amino acid residues
with aromatic side chains along the polypeptide chain (Spera et
al., J. Am. Chem. Soc., 113:5490-5492 (1991); Wishart et al.,
Biochemistry, 31:1647-1651 (1992), which are hereby incorporated by
reference in their entirety).
[0109] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D H,C,C,H-COSY NMR experiment or a RD 3D
H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons of amino acid residue i,
.sup.1H.sup.ali.sub.i, and aliphatic carbons of amino acid residue
i, .sup.13C.sup.ali.sub.i, of amino acid residue i. Then,
sequential assignments of the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, the chemical
shift values of a .gamma.-proton, .sup.1H.sup..gamma..sub.i, and a
.gamma.-carbon, .sup.13C.sup..gamma..sub.i, in particular, are
obtained by (i) matching the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i measured
using the RD 3D H,C,C,H-COSY NMR experiment or the RD 3D
H,C,C,H-TOCSY RD NMR experiment with the chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha., and
.sup.13C.sup..alpha. measured by the RD
3DH.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment
and the RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR
experiment, and (ii) using the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, including the
chemical shift values of .sup.1H.sup..gamma..sub.i and
.sup.13C.sup..epsilon..sub.i, to identify the type of amino acid
residue i.
[0110] A further aspect of the present invention involves a method
for sequentially assigning the chemical shift values of aliphatic
protons, .sup.1H.sup.ali, aliphatic carbons, .sup.13C.sup.ali, a
polypeptide backbone amide nitrogen, .sup.15N, and a polypeptide
backbone amide proton, .sup.1H.sup.N, of a protein molecule. The
method involves providing a protein sample and conducting a set of
reduced dimensionality (RD) nuclear magnetic resonance (NMR)
experiments on the protein sample, where the chemical shift values
of .sup.1H.sup.ali and .sup.13C.sup.ali which are encoded in peak
pairs of a 3D NMR spectrum are detected in a phase sensitive
manner, including: (1) a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment
to measure and connect the chemical shift values of the aliphatic
protons of amino acid residue i-1, .sup.1H.sup.ali.sub.i-1, the
aliphatic carbons of amino acid residue i-1,
.sup.13C.sup.ali.sub.i-1, the polypeptide backbone amide nitrogen
of amino acid residue i, .sup.15N.sub.i, and the polypeptide
backbone amide proton of amino acid residue i, .sup.15H.sup.N.sub.i
and (2) a RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR
experiment to measure and connect the chemical shift values of the
.beta.-proton of amino acid residue i, .sup.1H.sup..beta..sub.i,
the .beta.-carbon of amino acid residue i,
.sup.13C.sup..beta..sub.i, the .alpha.-proton of amino acid residue
i, .sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid
residue i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup.ali, .sup.13C.sup.ali, .sup.15N, and
.sup.1H.sup.N are obtained by (i) matching the chemical shift
values of the .alpha.- and .beta.-protons of amino acid residue
i-1, .sup.1H.sup..alpha./.beta..sub.i-1 and the .alpha.- and
.beta.-carbons of amino acid residue i-1,
.sup.13C.sup..alpha./.beta., with the chemical shift values of
.sup.1H.sup..alpha./.beta..sub.i and
.sup.13C.sup..alpha./.beta..sub.i of amino acid residue i, (ii)
using the chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.1-1 to identify the type of amino acid residue
i-1 (Wuthrich, NMR of Proteins and Nucleic Acids, Wiley, New York
(1986); Grzesiek et al., J. Biomol NMR, 3: 185-204 (1993), which
are hereby incorporated by reference in their entirety), and (iii)
mapping sets of sequentially connected chemical shift values to the
amino acid sequence of the polypeptide chain and using the chemical
shift values to locate secondary structure elements within the
polypeptide chain (Spera et al., J. Am. Chem. Soc., 113:5490-5492
(1991); Wishart et al., Biochemistry, 31:1647-1651 (1992), which
are hereby incorporated by reference in their entirety).
[0111] In one embodiment, the protein sample could, in addition to
the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD
3H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i. Then, sequential assignments of the chemical shift
value of .sup.13C'.sub.i are obtained by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1.sub.H.sup.N measured by
the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment.
[0112] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D HNN<CO,CA> NMR experiment to
measure and connect the chemical shift values of a polypeptide
backbone carbonyl carbon of amino acid residue i-1,
.sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift value of .sup.13C'.sub.i-1 are obtained by matching the
chemical shift value of .sup.13C.sup..alpha..sub.i measured by the
RD 3D HNN<CO,CA> NMR experiment with the sequentially
assigned chemical shift values of .sup.13C.sup..alpha., .sup.15N,
and .sup.1H.sup.N measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment.
[0113] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to (i) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta., CO,HA NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i, and (ii) a RD 3D HNN<CO,CA> NMR experiment
to measure and connect the chemical shift values of
.sup.13C'.sub.i, the .alpha.-carbon of amino acid residue i+1,
.sup.13C.sup..alpha..sub.i+1, the polypeptide backbone amide
nitrogen of amino acid residue i+1, .sup.15N.sub.i+1, and the
polypeptide backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1. Then, sequential assignments are obtained by
matching the chemical shift value of .sup.13C'.sub.i, measured by
the RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment.
[0114] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment (i)
to measure and connect the chemical shift values of
.sup.1H.sup..alpha./.beta..sub.i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i, and (ii) to identify NMR signals for
.sup.1H.sup..alpha./.beta..sub.i-1,
.sup.13C.sup..alpha./.beta..sub.i-1, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i in the RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment.
[0115] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D HA,CA,(CO),N,HN NMR experiment (i) to
measure and connect chemical shift values of
.sup.1H.sup..alpha..sub.i-1, .sup.13C.sup..alpha..sub.i-1,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i and (ii) to identify NMR
signals for .sup.1H.sup..alpha. and .sup.13C.sup..alpha. in the RD
3D HC,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment.
[0116] In another embodiment, the protein sample could, in addition
to the RD 3D HC,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a 3D HNNCACB NMR experiment to measure and
connect the chemical shift value of .sup.13C.sup..beta..sub.i,
.sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments are obtained by
matching the chemical shift values of .sup.13C.sup..beta..sub.i and
.sup.13C.sup..alpha..sub.i measured by said 3D HNNCACB NMR
experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
[0117] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 2D HB,CB,(CG,CD),HD NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i, and a
.delta.-proton of amino acid residue i with an aromatic side chain,
.sup.1H.sup..delta..sub.i. Then, sequential assignments are
obtained by matching the chemical shift values of
.sup.1H.sup..beta..sub.i and .sup.13C.sup..beta..sub.i measured by
said RD 2D HB,CB,(CG,CD)ND NMR experiment with the chemical shift
values of .sup.1H.sup..beta. and .sup.13C.sup..beta. measured by
the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR
experiment and the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment,
using the chemical shift values to identify amino acid residue i as
having an aromatic side chain, and mapping sets of sequentially
connected chemical shift values to the amino acid sequence of the
polypeptide chain and locating amino acid residues with aromatic
side chains along the polypeptide chain.
[0118] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment, be
further subjected to a RD 3D H,C,C,H-COSY NMR experiment or a RD 3D
H,C,C,H-TOCSY NMR experiment to measure and connect the chemical
shift values of aliphatic protons of amino acid residue i,
.sup.1H.sup.ali.sub.i, and aliphatic carbons of amino acid residue
i, .sup.13C.sup.ali.sub.i. Then, sequential assignments of the
chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i, the chemical shift values of a
.gamma.-proton, .sup.1H.sup..gamma..sub.i, and a .gamma.-carbon,
.sup.13C.sup..gamma..sub.i, in particular, are obtained by (i)
matching the chemical shift values of .sup.1H.sup.ali.sub.i and
.sup.13C.sup.ali.sub.i measured using the RD 3D H,C,C,H-COSY NMR
experiment or the RD 3D H,C,C,H-TOCSY NMR experiment with the
chemical shift values of .sup.1H.sup.ali and .sup.13C.sup.ali
measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and RD
3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment,
and (ii) using the chemical shift values of .sup.1H.sup.ali.sub.i
and .sup.13C.sup.ali.sub.i, including the chemical shift values of
.sup.1H.sup..gamma..sub.i and .sup.13C.sup..gamma..sub.i, to
identify the type of amino acid residue i.
[0119] The present invention also relates to a method for
sequentially assigning chemical shift values of aliphatic protons,
.sup.1H.sup.ali, aliphatic carbons, .sup.13C.sup.ali, a polypeptide
backbone amide nitrogen, .sup.15N, and a polypeptide backbone amide
proton, .sup.1H.sup.N, of a protein molecule. The method involves
providing a protein sample and conducting a set of reduced
dimensionality (RD) nuclear magnetic resonance (NMR) experiments on
the protein sample, where the chemical shift values of
.sup.1H.sup.ali and .sup.13C.sup.ali which are encoded in peak
pairs of a 3D NMR spectrum are detected in a phase sensitive
manner, including: (1) a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment
to measure and connect the chemical shift values of the aliphatic
protons of amino acid residue i-1, .sup.1H.sup.ali.sub.i-1, the
aliphatic carbons of amino acid residue i-1,
.sup.13C.sup.ali.sub.i-1, the polypeptide backbone amide nitrogen
of amino acid residue i, .sup.15N.sub.i, and the polypeptide
backbone amide proton of amino acid residue i, .sup.1H.sup.N.sub.i
and (2) a RD 3D HNNCAHA NMR experiment to measure and connect the
chemical shift values of the .alpha.-proton of amino acid residue
i, .sup.1H.sup..alpha..sub.i, the .alpha.-carbon of amino acid
residue i, .sup.13C.sup..alpha..sub.i, .sup.15N.sub.i, and
.sup.1H.sup.N.sub.i. Then, sequential assignments of the chemical
shift values of .sup.1H.sup.ali, .sup.13C.sup.ali, .sup.15N, and
.sup.1H.sup.N are obtained by (i) matching the chemical shift
values of the .alpha.-proton of amino acid residue i-1,
.sup.1H.sup..alpha..sub.i-1 and the .alpha.-carbon of amino acid
residue i-1, .sup.13C.sup..alpha..sub.i-1 with the chemical shift
values of .sup.1H.sup..alpha..sub.i and .sup.13C.sup..alpha..sub.i,
(ii) using the chemical shift values of .sup.1H.sup.ali.sub.i-1 and
.sup.13C.sup.ali.sub.1-1 to identify the type of amino acid residue
i-1 (Wuthrich, NMR of Proteins and Nucleic Acids, Wiley, New York
(1986); Grzesiek et al., J. Biomol. NMR, 3: 185-204 (1993), which
are hereby incorporated by reference in their entirety), and (iii)
mapping sets of sequentially connected chemical shift values to the
amino acid sequence of the polypeptide chain and using the chemical
shift values to locate secondary structure elements within the
polypeptide chain (Spera et al., J. Am. Chem. Soc., 113:5490-5492
(1991); Wishart et al., Biochemistry, 31:1647-1651 (1992), which
are hereby incorporated by reference in their entirety).
[0120] In one embodiment, the protein sample could, in addition to
the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment to
measure and connect the chemical shift values of a .beta.-proton of
amino acid residue i, .sup.1H.sup..beta..sub.i, a .beta.-carbon of
amino acid residue i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i, and a
polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i. Then, sequential assignments of the chemical shift
value of .sup.13C'.sub.i are obtained by matching the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, .sup.1H.sup..alpha..sub.i, and
.sup.13C.sup..alpha..sub.i measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment with
the sequentially assigned chemical shift values of
.sup.1H.sup..beta., .sup.13C.sup..beta., .sup.1H.sup..alpha.,
.sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by the
RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D HNNCAHA
NMR experiment.
[0121] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a RD 3D
HNN<CO,CA> NMR experiment to measure and connect the chemical
shift values of a polypeptide backbone carbonyl carbon of amino
acid residue i-1, .sup.13C'.sub.i-1, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i. Then, sequential
assignments of the chemical shift value of .sup.13C'.sub.i-1 are
obtained by matching the chemical shift value of
.sup.13C.sup..alpha..sub.i measured by the RD 3D HNN<CO,CA>
NMR experiment with the sequentially assigned chemical shift values
of .sup.13C.sup..alpha., .sup.15N, and .sup.1H.sup.N measured by
the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment.
[0122] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to (i) a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta., CO,HA NMR experiment to
measure and connect the chemical shift values of a .beta.-proton of
amino acid residue i, .sup.1H.sup..beta..sub.i, a .beta.-carbon of
amino acid residue i, .sup.13C.sup..beta..sub.i, the .alpha.-proton
of amino acid residue i, .sup.1H.sup..alpha..sub.i, the
.alpha.-carbon of amino acid residue i, .sup.13C.sup..alpha..sub.i,
and a polypeptide backbone carbonyl carbon of amino acid residue i,
.sup.13C'.sub.i, and (ii) a RD 3D HNN<CO,CA> NMR experiment
to measure and connect the chemical shift values of
.sup.13C'.sub.i, an .alpha.-carbon of amino acid residue i+1,
.sup.13C.sup..alpha..sub.i-1, a polypeptide backbone amide nitrogen
of amino acid residue i+1, .sup.15N.sub.i+1, and the polypeptide
backbone amide proton of amino acid residue i+1,
.sup.1H.sup.N.sub.i+1. Then, sequential assignments are obtained by
matching the chemical shift value of .sup.13C'.sub.i measured by
the RD 3D HNN<CO,CA> NMR experiment with the chemical shift
value of .sup.13C'.sub.i, measured by the RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,CO,HA NMR experiment.
[0123] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment (i)
to measure and connect the chemical shift values of the .alpha.-
and .beta.-protons of amino acid residue i-1,
.sup.1H.sup..alpha./.beta..sub.i-1, the .alpha.- and .beta.-carbons
of amino acid residue i-1, .sup.13C.sup..alpha./.beta..sub.i-1,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i, and (ii) to distinguish
NMR signals for the chemical shift values of
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1,
.sup.1H.sup..alpha..sub.i-1, and .sup.13C.sup..alpha..sub.i-1
measured by the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment from
NMR signals for the chemical shift values of
.sup.1H.sup.ali.sub.i-1 and .sup.13C.sup.ali.sub.i-1 other than
.sup.1H.sup..alpha./.beta..sub.i-1 and
.sup.13C.sup..alpha./.beta..sub.i-1 measured by the RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment.
[0124] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR experiment to
measure and connect the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i. Then, sequential
assignments are obtained by matching the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i measured
by said RD 3D H.sup..alpha./.beta.,C.sup..alpha./.beta.,N,HN NMR
experiment with the chemical shift values of
.sup.1H.sup..beta..sub.i-1, .sup.13C.sup..beta..sub.i-1,
.sup.1H.sup..alpha..sub.i-1, and .sup.13C.sup..alpha..sub.i-1
measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
[0125] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a 3D HNNCACB NMR
experiment to measure and connect the chemical shift values of
.sup.13C.sup..beta..sub.i, .sup.13C.sup..alpha..sub.i,
.sup.15N.sub.i, and .sup.1H.sup.N.sub.i. Then, sequential
assignments are obtained by matching the chemical shift values of
.sup.13C.sup..beta..sub.i and .sup.13C.sup..alpha..sub.i measured
by said 3D HNNCACB NMR experiment with the chemical shift values of
.sup.13C.sup..beta..sub.i-1 and .sup.13C.sup..alpha..sub.i-1
measured by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment.
[0126] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a RD 2D
HB,CB,(CG,CD),HD NMR experiment to measure and connect the chemical
shift values of .sup.1H.sup..beta..sub.i,
.sup.13C.sup..beta..sub.i, and a .delta.-proton of amino acid
residue i with an aromatic side chain, .sup.1H.sup..delta..sub.i.
Then, sequential assignments are obtained by matching the chemical
shift values of .sup.1H.sup..beta..sub.i and
.sup.13C.sup..beta..sub.i measured by said RD 2D HB,CB,(CG,CD),HD
NMR experiment with the chemical shift values of .sup.1H.sup..beta.
and .sup.13C.sup..beta. measured by the RD 3D H,C,(C-TOCSY-CO),N,HN
NMR experiment, using the chemical shift values to identify amino
acid residue i as having an aromatic side chain, and mapping sets
of sequentially connected chemical shift values to the amino acid
sequence of the polypeptide chain and by locating amino acid
residues with aromatic side chains along the polypeptide chain.
[0127] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, be further subjected to a RD 3D
H,C,C,H-COSY NMR experiment or a RD 3D H,C,C,H-TOCSY NMR experiment
to measure and connect the chemical shift values of aliphatic
protons of amino acid residue i, .sup.1H.sup.ali.sub.i, and
aliphatic carbons of amino acid residue i, .sup.13C.sup.ali.sub.i.
Then, sequential assignments of the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, the chemical
shift values of a .gamma.-proton, .sup.1H.sup..gamma..sub.i, and a
.gamma.-carbon, .sup.13C.sup..gamma..sub.i, in particular, are
obtained by (i) matching the chemical shift values of
.sup.1H.sup.ali and .sup.13C.sup.ali measured using the RD 3D
H,C,C,H-COSY NMR experiment or the RD 3D H,C,C,H-TOCSY NMR
experiment with the chemical shift values of
.sup.1H.sup..beta..sub.i, .sup.13C.sup..beta..sub.i,
.sup.1H.sup..alpha..sub.i, and .sup.13C.sup..alpha..sub.i measured
by the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and the RD 3D
HNNCAHA NMR experiment, and (ii) using the chemical shift values of
.sup.1H.sup.ali.sub.i and .sup.13C.sup.ali.sub.i, including the
chemical shift values of .sup.1H.sup..gamma..sub.i, and
.sup.13C.sup..gamma..sub.i, to identify the type of amino acid
residue i.
[0128] Another aspect of the present invention involves a method
for obtaining nearly complete assignments of chemical shift values
of .sup.1H, .sup.13C and .sup.15N of a protein molecule (excluding
only chemical shift values of .sup.13C.sup..delta. and
.sup.15N.sup..epsilon.2 of glutamines, of .sup.13C.sup..gamma. and
.sup.15N.sup..delta.2 of asparagines, of .sup.13C.sup..epsilon.3,
.sup.1H.sup..epsilon.3, .sup.13C.sup..zeta.2, .sup.1H.sup..zeta.2,
.sup.13C.sup..zeta.3, .sup.1H.sup..zeta.3, .sup.13C.sup..eta.2, and
.sup.1H.sup..eta.2 groups of tryptophans, of .sup.13C.sup..epsilon.
and .sup.1H.sup..epsilon. of methionines, and of labile sidechain
protons that exchange rapidly with the protons of the solvent
water) (Yamazaki et al., J. Am. Chem. Soc., 115:11054-11055 (1993),
which is hereby incorporated by reference in its entirety), which
are required for the determination of the tertiary structure of a
protein in solution (Wuthrich, NMR of Proteins and Nucleic Acids,
Wiley, New York (1986), which is hereby incorporated by reference
in its entirety). The method involves providing a protein sample
and conducting four reduced dimensionality (RD) nuclear magnetic
resonance (NMR) experiments on the protein sample, where the
chemical shift values of .sup.1H and .sup.13C which are encoded in
peak pairs of a 3D NMR spectrum are detected in a phase sensitive
manner and (1) a first experiment is selected from a RD
three-dimensional (3D)
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment, a
RD 3D HA,CA,(CO),N,HN NMR experiment, or a RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment for obtaining sequential
correlations of chemical shift values; (2) a second experiment is
selected from a RD 3D HNNCAHA NMR experiment, a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, or a
RD 3D HNN<CO,CA> NMR experiment for obtaining intraresidue
correlations of chemical shift values; (3) a third experiment is a
RD 3D H,C,C,H-COSY NMR experiment for obtaining assignments of
aliphatic and aromatic sidechain chemical shift values; and (4) a
fourth experiment is a RD 2D HB,CB,(CG,CD),HD NMR experiment for
obtaining assignments of aromatic sidechain chemical shift
values.
[0129] In one embodiment of this method, the protein sample could
be further subjected to a RD 2D H,C,H-COSY NMR experiment for
obtaining assignments of aliphatic and aromatic sidechain chemical
shift values.
[0130] In another embodiment of this method, the first experiment
is the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment and the second experiment is the RD 3D HNNCAHA NMR
experiment.
[0131] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D HA,CA,(CO),N,HN NMR experiment to distinguish
between NMR signals for .sup.1H.sup..alpha./.sup.13C.sup..alpha.
and .sup.1H.sup..beta./.sup.13C.sup..beta. from the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment.
[0132] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment to obtain
assignments of chemical shift values of .sup.1H.sup.ali and
.sup.13C.sup.ali.
[0133] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN
NMR experiment to obtain assignments of chemical shift values of
.sup.1H.sup..beta. and .sup.13C.sup..beta..
[0134] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D HNN<CO,CA> NMR experiment to obtain
assignments of chemical shift values of polypeptide backbone
carbonyl carbons, .sup.13C'.
[0135] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,
CO,HA NMR experiment to obtain assignments of chemical shift values
of polypeptide backbone carbonyl carbons, .sup.13C'.
[0136] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D HNN<CO,CA> NMR experiment and a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
obtain assignments of chemical shift values of .sup.13C'.
[0137] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D H,C,C,H-TOCSY NMR experiment to obtain
assignments of chemical shift values of .sup.1H and .sup.13C of
aliphatic sidechains.
[0138] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a RD 3D H,C,C,H-TOCSY NMR experiment to obtain
assignments of chemical shift values of .sup.1H and .sup.13C of
aromatic sidechains.
[0139] In another embodiment, the protein sample could, in addition
to the RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment, RD 3D HNNCAHA NMR experiment, RD 3D H,C,C,H-COSY NMR
experiment, and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further
subjected to a 3D HNNCACB NMR experiment to obtain assignments of
chemical shift values of .sup.13C.sup..beta..
[0140] In yet another embodiment of this method, the first
experiment is the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and
the second experiment is the RD 3D HNNCAHA NMR experiment.
[0141] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
HA,CA,(CO),N,HN NMR experiment to identify NMR signals for
.sup.1H.sup..alpha./.sup.13C.sup..alpha. in the RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment.
[0142] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment to
obtain assignments of chemical shift values of .sup.1H.sup..beta.
and .sup.13C.sup..beta..
[0143] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
HNN<CO,CA> NMR experiment to obtain assignments of chemical
shift values of polypeptide backbone carbonyl carbons,
.sup.13C'.
[0144] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta., CO,HA NMR experiment to
obtain assignments of chemical shift values of polypeptide backbone
carbonyl carbons, .sup.13C'.
[0145] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
HNN<CO,CA> NMR experiment and a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
obtain assignments of chemical shift values of .sup.13C'.
[0146] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
H,C,C,H-TOCSY NMR experiment to obtain assignments of chemical
shift values of .sup.1H and .sup.13C of aliphatic sidechains.
[0147] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a RD 3D
H,C,C,H-TOCSY NMR experiment to obtain assignments of chemical
shift values of .sup.1H and .sup.13C of aromatic sidechains.
[0148] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D HNNCAHA
NMR experiment, RD 3D H,C,C,H-COSY NMR experiment, and RD 2D
HB,CB,(CG,CD),HD NMR experiment, be further subjected to a 3D
HNNCACB NMR experiment to obtain assignments of chemical shift
values of .sup.13C.sup..beta..
[0149] In yet another embodiment of this method, the first
experiment is the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and
the second experiment is the RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment.
[0150] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D HA,CA,(CO),N,HN NMR
experiment to identify NMR signals for .sup.1H.sup..alpha. and
.sup.13C.sup..alpha. in the RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment.
[0151] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR experiment to
identify NMR signals for .sup.1H.sup..alpha./.beta. and
.sup.13C.sup..alpha./.beta. in the RD 3D H,C,(C-TOCSY-CO),N,HN NMR
experiment.
[0152] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D HNN<CO,CA> NMR
experiment to obtain assignments of chemical shift values of
polypeptide backbone carbonyl carbons, .sup.13C'.
[0153] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
obtain assignments of chemical shift values of polypeptide backbone
carbonyl carbons, .sup.13C'.
[0154] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D HNN<CO,CA> NMR
experiment and a RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR experiment to
obtain assignments of chemical shift values of .sup.13C'.
[0155] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D H,C,C,H-TOCSY NMR
experiment to obtain assignments of chemical shift values of
.sup.1H and .sup.13C of aliphatic sidechains.
[0156] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a RD 3D H,C,C,H-TOCSY NMR
experiment to obtain assignments of chemical shift values of
.sup.1H and .sup.13C of aromatic sidechains.
[0157] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.,N,HN NMR experiment, RD 3D
H,C,C,H-COSY NMR experiment, and RD 2D HB,CB,(CG,CD),HD NMR
experiment, be further subjected to a 3D HNNCACB NMR experiment to
obtain assignments of chemical shift values of
.sup.13C.sup..beta..
[0158] In yet another embodiment of this method, the first
experiment is the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment and
the second experiment is the RD 3D HNN<CO,CA> NMR
experiment.
[0159] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
HNN<CO,CA> NMR experiment, RD 3D H,C,C,H-COSY NMR experiment,
and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further subjected to
a RD 3D HA,CA,(CO),N,HN NMR experiment to identify NMR signals for
.sup.1H.sup..alpha. and .sup.13C.sup..alpha. in the RD 3D
H,C,(C-TOCSY-CO),N,HN NMR experiment.
[0160] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
HNN<CO,CA> NMR experiment, RD 3D H,C,C,H-COSY NMR experiment,
and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further subjected to
a RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN NMR
experiment to identify NMR signals for .sup.1H.sup..alpha./.beta.
and .sup.13C.sup..alpha./.beta. in the RD 3D H,C,(C-TOCSY-CO),N,HN
NMR experiment.
[0161] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
HNN<CO,CA> NMR experiment, RD 3D H,C,C,H-COSY NMR experiment,
and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further subjected to
a RD 3D H.sup..alpha./.beta.C.sup..alpha./.beta.,CO,HA NMR
experiment to obtain assignments of chemical shift values of
polypeptide backbone carbonyl carbons, .sup.13C'.
[0162] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
HNN<CO,CA> NMR experiment, RD 3D H,C,C,H-COSY NMR experiment,
and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further subjected to
a RD 3D H,C,C,H-TOCSY NMR experiment to obtain assignments of
chemical shift values of .sup.1H and .sup.13C of aliphatic
sidechains.
[0163] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
HNN<CO,CA> NMR experiment, RD 3D H,C,C,H-COSY NMR experiment,
and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further subjected to
a RD 3D H,C,C,H-TOCSY NMR experiment to obtain assignments of
chemical shift values of .sup.1H and .sup.13C of aromatic
sidechains.
[0164] In another embodiment, the protein sample could, in addition
to the RD 3D H,C,(C-TOCSY-CO),N,HN NMR experiment, RD 3D
HNN<CO,CA> NMR experiment, RD 3D H,C,C,H-COSY NMR experiment,
and RD 2D HB,CB,(CG,CD),HD NMR experiment, be further subjected to
a 3D HNNCACB NMR experiment to obtain assignments of chemical shift
values of .sup.13C.sup..beta..
[0165] In addition, the above-described method for obtaining
assignments of chemical shift values of .sup.1H, .sup.13C and
.sup.15N of a protein molecule can involve further subjecting the
protein sample to nuclear Overhauser effect spectroscopy (NOESY)
(Wuthrich, NMR of Proteins and Nucleic Acids, Wiley, New York
(1986), which is hereby incorporated by reference in its entirety),
to NMR experiments that measure scalar coupling constants
(Eberstadt et al., Angew Chem. Int. Ed. Engl., 34:1671-1695 (1995);
Cordier et al., J. Am. Chem. Soc., 121:1601-1602 (1999), which are
hereby incorporated by reference in their entirety), or to NMR
experiments that measure residual dipolar coupling constants
(Prestegard, Nature Struct. Biol., 5:517-522 (1998); Tjandra et
al., Science, 278:1111-1114 (1997), which are hereby incorporated
by reference in their entirety), to deduce the tertiary fold or
tertiary structure of the protein molecule.
[0166] A standard set of experiments (3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN, 3D HNNCAHA, 3D
HH.sup..alpha./.beta.C.sup..alpha./.beta.COHA, 3D HNNCACB, 3D
HNN<CO,CA>, 3D HCCH-COSY, 3D HCCH-TOCSY, 2D HBCB(CDCG)HD, and
2D .sup.1H-TOCSY-relayed HCH-COSY) can be employed for obtaining
nearly complete resonance assignments of proteins including
aliphatic and aromatic side chain spin systems.
[0167] For larger proteins, complementary recording of highly
sensitive 3D HACA(CO)NHN promises (i) to yield spin systems which
escape detection in
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN, and (ii) to offer
the distinction of .alpha.- and .beta.-moiety resonances by
comparison with H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN.
Furthermore, employment of 50% random fractional protein
deuteration (LeMaster, Annu. Rev. Biophys. Biophys Chem., 19:43-266
(1990); Nietlispach et al., J. Am. Chem. Soc., 118:407-415 (1996);
Shan et al., J. Am. Chem. Soc., 118:6570-6579 (1996); Leiting et
al., Anal. Biochem., 265:351-355 (1998); Hochuli et al., J. Biomol.
NMR, 17:33-42 (2000), which are hereby incorporated by reference in
their entirety) in combination with the standard suite of NMR
experiments (or transverse relaxation-optimized spectroscopy
(TROSY) versions thereof) is attractive. The impact of deuteration
for recording 4D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN
for proteins reorienting with correlation times up to around 20 ns
(corresponding to a molecular weight around 30 kDa at ambient T)
has been demonstrated (Nietlispach et al., J. Am. Chem. Soc.,
118:407-415 (1996), which is hereby incorporated by reference in
its entirety). Accordingly, 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN can be expected to
maintain its pivotal role for obtaining complete resonance
assignments for deuterated proteins at least up to about that size.
Furthermore, protein deuteration offers the advantage that HNNCACB,
which can be expected to become significantly less sensitive than
HNNCAHA for larger non-deuterated systems, (Szyperski et al., J.
Biomol. NMR, 11:387-405 (1998), which is hereby incorporated by
reference in its entirety) can be kept to recruit
.sup.13C.sup..beta. chemical shifts for sequential assignment (Shan
et al., J. Am. Chem. Soc., 118:6570-6579 (1996), which is hereby
incorporated by reference in its entirety).
[0168] If solely chemical shifts are considered, the unambiguous
identification of peak pairs is more involved whenever multiple
peak pairs with degenerate chemical shifts in the other dimensions
are present. The acquisition of the corresponding central peaks
addresses this complication in a conceptually straightforward
fashion. However, it is important to note that pairs of peaks
generated by a chemical shift splitting have quite similar
intensity. In contrast, peak pairs arising from different moieties,
possible located in different amino acid residues, most often do
not show similar intensity. This is because the nuclear spin
relaxation times, which determine the peak intensities, vary within
each residue as well as along the polypeptide chain. One may thus
speak of a "nuclear spin relaxation time labeling" of peak pairs,
which makes their identification an obvious task in most cases.
[0169] Using cryogenic probes can reduce NMR measurement times by
about a factor of 10 or more (Flynn et al., J. Am. Chem. Soc.,
122:4823-4824 (2000), which is hereby incorporated by reference in
its entirety). The high sensitivity of cryogenic probes shifts even
the recording of RD NMR experiments entirely into the sampling
limited data acquisition regime. In view of this dramatic reduction
in spectrometer time demand, minimally achievable RD NMR
measurement times are of keen interest to be able to adapt the NMR
measurement times to sensitivity requirements in future HTP
endeavours. To further reduce the measurement time, and in view of
the aforementioned "spin relaxation time labelling" of peak pairs,
one may decide to also discard the use of .sup.13C-steady state
magnetization for central peak detection.
[0170] Although RD NMR was proposed in 1993 (Szyperski et al., J.
Biomol. NMR, 3:127-132 (1993); Szyperski et al., J. Am. Chem. Soc.,
115:9307-9308 (1993), which are hereby incorporated by reference in
their entirety), its wide-spread use has been delayed by the more
demanding spectral analysis when compared to conventional TR NMR.
In particular, the necessity to extract chemical shifts from
in-phase splittings suggests that strong computer support is key
for employment of RD NMR on a routine basis. This can be readily
addressed by using automated resonance assignment software for
automated analysis of RD TR NMR data.
[0171] In conclusion, the joint employment of RD NMR spectroscopy
(as well as phase-sensitively detected RD NMR spectroscopy),
cryogenic probes, and automated backbone resonance assignment will
allow one to determine a protein's backbone resonance assignments
and secondary structure in a short time.
EXAMPLES
[0172] The following examples are provided to illustrate
embodiments of the present invention but are by no means intended
to limit its scope.
Example 1
Sample Preparation
[0173] NMR measurements were performed using a 1 mM solution of
uniformly .sup.13C/.sup.15N enriched "Z-domain" of the
Staphylococcal protein A (Tashiro et al., J. Mol. Biol.,
272:573-590 (1997); Lyons et al., Biochemistry, 32:7839-7845
(1993), which are hereby incorporated by reference in their
entirety) dissolved in 90% D.sub.2O/10% H.sub.2O (20 M K--PO.sub.4)
at pH=6.5 and a 2 mM solution of .sup.15N/.sup.13C doubly labeled
ubiquitin in 90% H.sub.2O/10% D.sub.2O (50 mM K--PO.sub.4;
pH=5.8).
Example 2
NMR Spectroscopy
[0174] Multidimensional NMR experiments (FIG. 2; Table 1) were
recorded for a 1 mM solution of the 8.5 kDa Z-domain protein and/or
a 2 mM solution of ubiquitin at a temperature of 25.degree. C. The
spectra were assigned, and the chemical shifts obtained from RD NMR
were in very good agreement with those previously determined at
30.degree. C. using conventional triple resonance (TR) NMR
spectroscopy (Tashiro et al., J. Mol. Biol., 272:573-590 (1997);
Lyons et al., Biochemistry, 32:7839-7845 (1993), which are hereby
incorporated by reference in their entirety).
[0175] NMR experiments were recorded at a temperature of 25.degree.
C. on a Varian Inova 600 spectrometer equipped with a new
generation .sup.1H{.sup.13C,.sup.15N} triple resonance probe which
exhibits a signal-to-noise ratio of 1200:1 for a standard 0.1%
ethylbenzene sample.
[0176] Specific embodiments of the 11 RD NMR experiments disclosed
by the present invention were implemented for the present study.
FIG. 2 provides a survey of (i) the names, (ii) the magnetization
transfer pathways and (iii) the peak patterns observed in the
projected dimension of each of the 11 RD NMR experiments disclosed
by the present invention. For simplicity, FIG. 2 shows the in-phase
peak pattern from the cosine-modulated RD NMR experiments. When
acquiring the sine-modulated congeners, anti-phase peak pairs were
observed instead (see FIG. 1). The group comprising the first three
experiments are designed to yield "sequential" connectivities via
one-bond scalar couplings: 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN (FIG. 2A), 3D
HACA(CO)NHN (FIG. 2B), and 3D HC(C-TOCSY-CO)NHN (FIG. 2C). The
following three experiments provide "intraresidual" connectivities
via one-bond scalar couplings: 3D HNNCAHA (FIG. 2D), 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.COHA (FIG. 2E), and 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.NNH (FIG. 2F). 3D
HNN<CO,CA> (FIG. 2G) offers both intraresidual
.sup.1H.sup.N-.sup.13C.sup..alpha. and sequential
.sup.1H.sup.N-.sup.13C' connectivities. Although 3D HNNCAHA (FIG.
2D), 3D H.sup..alpha./.beta.C.sup..alpha./.beta.NHN (FIG. 2F) and
3D HNN<CO,CA> (FIG. 2G) also provide sequential
connectivities via two-bond
.sup.13C.sup..alpha..sub.i-1-.sup.15N.sub.i scalar couplings, those
are usually smaller than the one-bond couplings (Cavanagh et al.,
Protein NMR Spectroscopy, Academic Press, San Diego, (1996), which
is hereby incorporated by reference in its entirety), and obtaining
complete backbone resonance assignments critically depends on
experiments designed to provide sequential connectivities via
one-bond couplings (FIGS. 2D-F). 3D HCCH-COSY (FIG. 2H) and 3D
HCCH-TOCSY (FIG. 2I) allow one to obtain assignments for the
"aliphatic" side chain spin systems, while 2D HBCB(CDCG)HD (FIG.
2J) and 2D .sup.1H-TOCSY-relayed HCH-COSY (FIG. 2K) provide the
corresponding information for the "aromatic" spin systems.
[0177] The RD NMR experiments are grouped accordingly in Table 1,
which lists for each experiment (i) the nuclei for which the
chemical shifts are measured, (ii) if and how the central peaks are
acquired and (iii) additional notable technical features.
State-of-the art implementations (Cavanagh et al., Protein NMR
Spetroscopy, Academic Press, San Diego, (1996); Kay, J. Am. Chem.
Soc., 115:2055-2057 (1993); Grzesiek et al., J. Magn Reson.,
99:201-207 (1992); Montelione et al., J. Am. Chem. Soc.,
114:10974-10975 (1992); Boucher et al., J. Biomol. NMR, 2:631-637
(1992); Yamazaki et al., J. Am. Chem. Soc., 115:11054-11055 (1993);
Zerbe et al., J. Biomol. NMR, 7:99-106 (1996); Grzesiek et al., J.
Biomol. NMR 3:185-204 (1993), which are hereby incorporated by
reference in their entirety) making use of pulsed field z-gradients
for coherence selection and/or rejection, and sensitivity
enhancement (Cavanagh et al., Protein NMR Spectroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) were chosen, which allow executing these
experiments with a single transient per acquired free induction
decay (FID). Semi (Grzesiek et al., J. Biomol. NMR, 3:185-204
(1993), which is hereby incorporated by reference in its entirety)
constant-time (Cavanagh et al., Protein NMR Spectroscopy, Academic
Press, San Diego, (1996), which is hereby incorporated by reference
in its entirety) chemical shift frequency-labeling modules were
used throughout in the indirect dimensions in order to minimize
losses arising from transverse nuclear spin relaxation. FIGS. 3A-K
provide comprehensive descriptions of the RD NMR r.f. pulse
sequences.
[0178] In total, nine RD TR NMR experiments were recorded for the
Z-domain protein (Table 2) and eleven RD TR NMR experiments for
ubiquitin (Table 3). FIGS. 5-15 show composite plots of
[.omega..sub.1,.omega..sub.3]- or
[.omega..sub.1,.omega..sub.2]-strips taken from the recorded
phase-sensitively detected RD NMR experiments. Except for 3D
HNNCAHA, 3D HNN<CO,CA> and 2D .sup.1H-TOCSY-relayed HCH-COSY
(FIG. 1), central peaks were derived from .sup.13C magnetization
(FIG. 2; Table 1). Hence, two subspectra, I and II containing the
peak pairs and central peaks respectively, were generated
(Szyperski et al., J. Am. Chem. Soc., 118:8146-8147 (1996);
Szyperski et al., J. Biomol. NMR, 11:387-405 (1998), which are
hereby incorporated by reference in their entirety) for eight of
the RD NMR experiments (FIG. 2).
TABLE-US-00002 TABLE 2 Phase-Sensitively Detected RD NMR Data Sets
Recorded for "Z-domain" Protein 3D HACA(CO) 3D 3D
H.sup..alpha./.beta.C.sup..alpha./.sup..beta. 3D
H.sup..alpha./.beta.C.sup..alpha./.sup..beta. 3D NHN HNNCAHA
(CO)NHN NHN HNN<CO,CA> .sup.1H Resonance Frequency (MH.sub.z)
No. of Points.sup.a 600 600 600 600 600 collected 40, 28, 512 64,
28, 512 76, 28, 512 68, 28, 512 56, 28, 512 after LP 80, 56, 512
96, 56, 512 152, 56, 512 136, 56, 512 112, 56, 512 after zero
filling 256, 64, 1024 256, 64, 1024 256, 64, 1024 256, 64, 1024
256, 64, 1024 window functions.sup.b sin 70/70/90 sin 70/70/90 sin
70/70/90 sin 70/70/90 sin 70/70/90 No. of Transients.sup.c 2 2 2 2
2 Spectral Width.sup.d 6000 (RD), 5200.2 (RD), 12001.2 (RD),
12001.2 (RD), 8000 (RD), (.omega..sub.1, .omega..sub.2,
.omega..sub.3, .omega..sub.4; Hz) 1300, 6983 1300, 6983 1300, 6983
1300, 6983 1300, 6983 t.sub.max (ms).sup.e 6.5, 20.77, 12.11,
20.77, 6.25, 20.77, 5.58, 20.77, 6.88, 20.77, 73.18 73.18 73.18
73.18 73.18 Carrier Position 53.05 (3.78), 53.05 (3.78), 38.05
(3.78), 38.05 (3.78), 171.07 (53.0), (.omega..sub.1, .omega..sub.2,
.omega..sub.3, .omega..sub.4; ppm).sup.f 119.3, 4.78 119.3, 4.78
119.3, 4.78 119.3, 4.78 119.3, 4.78 Recycle Delay (s).sup.g 0.67
0.67 0.67 0.67 0.67 Collection Time (h).sup.h 6.7 10.73 12.74 11.4
9.38 3D HC(C- 2D TOCSY-- 3D HCCH-- 3D HCCH- HBCB(CGCD) CO)NHN COSY
TOCSY HD .sup.1H Resonance Frequency (MH.sub.z) No. of Points.sup.a
600 600 600 600 collected 76, 28, 512 90, 20, 12 80, 20, 512 76,
512 after LP 152, 56, 512 180, 40, 512 160, 40, 512 152, 512 after
zero filling 256, 64, 1024 256, 64, 1024 256, 64, 1024 512, 1024
window functions.sup.b sin 70/70/90 sin 70/70/90 sin 70/70/90 sin
70/90 No. of Transients.sup.c 2 2 2 64 Spectral Width.sup.d 12001.2
(RD), 12001.2 (RD), 12001.2 (RD), 12001.2 (RD), (.omega..sub.1,
.omega..sub.2, .omega..sub.3, .omega..sub.4; Hz) 1300, 6983 3125,
6983 3125, 6983 6983 t.sub.max (ms).sup.e 6.25, 20.77, 7.42, 6.08,
6.58, 6.08, 6.25, 73.18 73.18 73.18 73.18 Carrier Position 38.05
(3.78), 38.05 (3.78), 38.05 (3.78), 33.04(3.78), (.omega..sub.1,
.omega..sub.2, .omega..sub.3, .omega..sub.4; ppm).sup.f 119.3, 4.78
38.05, 4.78 38.05, 4.78 4.78 Recycle Delay (s).sup.g 0.67 0.67 0.77
0.72 Collection Time (h).sup.h 12.74 10.77 11.0 7.82 .sup.a"No. of
Points" represents the number of complex data points used to sample
indirect dimensions. Before Fourier transformation, the time domain
points are extended by linear prediction (LP). .sup.bThe "Window
Function" is a mathematical function multiplied with the FID along
each indirect dimension before zero-filling and Fourier
transformation. .sup.c"No. of Transients" represents the number of
FIDs acquired for each real increment. .sup.d"Spectral Width" is
the frequency range covered in each dimension. .sup.e"t.sub.max" is
the maximum chemical shift evolution time. .sup.f"Carrier Position"
refers to the frequency (in ppm) of the center point of the
spectrum along each dimension. .sup.g"Recycle Delay" denotes the
relaxation delay between acquisitions of FIDs. .sup.h"Collection
Time" is the total measurement time.
TABLE-US-00003 TABLE 3 Phase-Sensitively Detected RD NMR Data Sets
Recorded for Ubiquitin 3D 3D 3D HACA(CO) 3D
H.sup..alpha./.beta.C.sup..alpha./.beta. 3D
H.sup..alpha./.beta.C.sup..alpha./.sup..beta. HNN<CO, 3D
H.sup..alpha./.beta.C.sup..alpha./.sup..beta. NHN HNNCAHA (CO)NHN
NHN CA> COHA .sup.1H Resonance Frequency (MH.sub.z) No. of
Points.sup.a 600 600 600 600 600 600 collected 40, 28, 512 56, 28,
512 76, 28, 512 68, 28, 512 56, 28, 512 76, 32, 512 after LP 152,
64, 512 after zero filling 256, 64, 1024 window functions.sup.b sin
70/70/90 No. of Transients.sup.c 2 2 2 2 2 2 Spectral Width.sup.d
6000 (RD), 6000 (RD), 12001.2 (RD), 12001.2 (RD), 8000 (RD),
12001(RD), (.omega..sub.1, .omega..sub.2, .omega..sub.3,
.omega..sub.4; Hz) 1337, 7018 1337, 7018 1337, 7018 1337, 7018
1337, 7018 1800, 7018 t.sub.max (ms).sup.e 6.5, 20.2, 9.17, 20.2,
6.25, 20.2, 5.58, 20.2, 6.88, 20.2, 6.25, 17.2, 72.8 72.8 72.8 72.8
72.8 72.8 Carrier Position 53.04 (3.78), 53.04 (3.78), 38.05
(3.78), 38.05 (3.78), 171.07 (53.0), 38.05 (3.78), (.omega..sub.1,
.omega..sub.2, .omega..sub.3, .omega..sub.4; ppm).sup.f 119.3, 4.78
119.3, 4.78 119.3, 4.78 119.3, 4.78 119.3, 4.78 172.4, 4.78 Recycle
Delay (s).sup.g 0.67 0.67 0.67 0.67 0.67 0.67 Collection Time
(h).sup.h 6.7 9.38 12.73 11.39 9.38 14.55 3D HC(C- 2D 2D H- TOCSY--
3D HCCH-- 3D HCCH- HBCB(CGCD) TOCSY-- CO)NHN COSY TOCSY HD
HCH--COSY .sup.1H Resonance Frequency (MH.sub.z) No. of
Points.sup.a 600 600 600 600 600 collected 76, 28, 512 90, 20, 512
80, 20, 512 76, 512 100, 512 after LP 152, 512 150, 512 after zero
filling 512, 1024 512, 1024 window functions.sup.b sin 70/90 sin
70/90 No. of Transients.sup.c 2 2 2 32 16 Spectral Width.sup.d
12001.2 (RD), 12001.2 (RD), 12001.2 (RD), 12001(RD), 10000(RD),
(.omega..sub.1, .omega..sub.2, .omega..sub.3, .omega..sub.4; Hz)
1337, 7018 3125, 7018 3125, 7018 7018 7018 t.sub.max (ms).sup.e
6.25, 20.2, 7.42, 6.08, 6.58, 6.08, 6.25, 72.8 9.9, 72.8 72.8 72.8
72.8 Carrier Position 38.05 (3.78), 38.05(3.78), 38.05 (3.78),
33.05 (3.78), 120.05 (4.78), (.omega..sub.1, .omega..sub.2,
.omega..sub.3, .omega..sub.4; ppm).sup.f 119.3, 4.78 38.05,4.78
38.05, 4.78 4.78 4.78 Recycle Delay (s).sup.g 0.67 0.67 0.77 0.72
0.77 Collection Time (h).sup.h 12.73 10.77 10.99 3.91 1.37
.sup.a"No. of Points" represents the number of complex data points
used to sample indirect dimensions. Before Fourier transformation,
the time domain points are extended by linear prediction (LP).
.sup.bThe "Window Function" is a mathematical function multiplied
with the FID along each indirect dimension before zero-filling and
Fourier transformation. .sup.c"No. of Transients" represents the
number of FIDs acquired for each real increment. .sup.d"Spectral
Width" is the frequency range covered in each dimension.
.sup.e"t.sub.max" is the maximum chemical shift evolution time.
.sup.f"Carrier Position" refers to the frequency (in ppm) of the
center point of the spectrum along each dimension. .sup.g"Recycle
Delay" denotes the relaxation delay between acquisitions of FIDs.
.sup.h"Collection Time" is the total measurement time.
Example 3
HTP Assignment Strategy
[0179] The comprehensive analysis of the suite of multidimensional
spectra recorded for the present study (Tables 1-3) lays the
foundation to devise strategies for RD NMR-based HTP resonance
assignment of proteins.
[0180] For proteins in the molecular weight range up to about 20
kDa, 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN plays a
pivotal role. Firstly, the peak patterns observed along
.omega..sub.1(.sup.13C.sup..alpha./.beta.) in subspectra I and II
enable sequential resonance assignment in combination with HNNCAHA
and HNNCACB, respectively, by matching intraresidue and sequential
.sup.1H.sup..alpha., .sup.13C.sup..alpha. and .sup.13C.sup..beta.
chemical shifts. (When considering "nuclear spin relaxation time
labelling" of peak pairs, subspectrum II derived from .sup.13C
steady state magnetization provides largely redundant information
when compared with subspectrum I. However, the observation of the
central peaks allows direct matching of peak positions between
subspectrum II, essentially a CBCA(CO)NHN spectrum, and HNNCACB.)
Moreover, this set of chemical shifts alone provides valuable
information for amino acid type identification (Zimmerman et al.,
J. Mol. Biol., 269:592-610 (1997); Cavanagh et al., Protein NMR
Spetroscopy, Academic Press, San Diego, (1996); Grzesiek et al., J.
Biomol NMR, 3:185-204 (1993), which are hereby incorporated by
reference in their entirety). Complementary recording of 3D
H.sup..alpha./.beta.C.sup..alpha./.beta.COHA and 3D
HNN<CO,CA> contributes polypeptide backbone .sup.13C.dbd.O
chemical shift measurements for establishing sequential
assignments: the intraresidue correlation is obtained by
.omega..sub.1(.sup.13C.sup..alpha./.beta.) peak pattern matching
with 3D H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN, and the
sequential correlation is inferred from .sup.13C.sup..alpha.,
.sup.15N and .sup.1H.sup.N chemical shifts in 3D HNN<CO,CA>
(Szyperski et al., J. Biomol. NMR, 11:387-405 (1998), which is
hereby incorporated by reference in its entirety). Notably, even
for medium-sized (non-deuterated) proteins this approach is
superior to the use of a low sensitivity HNNCACO-type experiment
(e.g., in combination with HNNCOCA), where the magnetization
transfer via rapidly relaxing .sup.13C.sup..alpha. relies on the
rather small .sup.15N--.sup.13C.sup..alpha. one-bond scalar
coupling. Secondly, comparison of
.omega..sub.1(.sup.13C.sup..alpha./.beta.) peak patterns with 3D
HCCH-COSY and TOCSY connects the
C.sup..alpha./.beta./H.sup..alpha./.beta. chemical shifts with
those of the aliphatic side chain spin systems (For Z-domain,
complete side chain assignments were obtained for all but six
residues using 3D HCCH-COSY only.), while comparison of
.omega..sub.1(.sup.13C.sup..beta.) peaks with 2D HBCB(CDCG)HD and
subsequent linking with .sup.1H.sup..delta. chemical shifts
detected in 2D .sup.1H-TOCSY-relayed HCH-COSY affords assignment of
the aromatic spin systems. Since for many amino acid residues the
two .beta.-protons exhibit non-degenerate chemical shifts, the
connection of H.sup..alpha./.beta.C.sup..alpha./.beta.(CO)NHN and
HBCB(CDCG)HD or HCCH-COSY/TOCSY may in fact often rely on
comparison of three chemical shifts, i.e.,
.delta.(.sup.1H.sup..beta.2), .delta.(.sup.1H.sup..beta.3) and
.delta.(.sup.13C.sup..beta.). This consideration underscores the
potential of recruiting .beta.-proton chemical shifts for
establishing sequential resonance assignments.
[0181] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
* * * * *