U.S. patent application number 11/320157 was filed with the patent office on 2006-07-27 for sea-trosy and related methods.
This patent application is currently assigned to Triad Therapeutics, Inc.. Invention is credited to Maurizio Pellecchia, Daniel S. Sem.
Application Number | 20060166279 11/320157 |
Document ID | / |
Family ID | 26946760 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166279 |
Kind Code |
A1 |
Sem; Daniel S. ; et
al. |
July 27, 2006 |
Sea-trosy and related methods
Abstract
A method for preferentially observing an exposed position (1c)
of a macromolecule. A sample is obtained having a macromolecule
(1a) with a first proton (1) and a second molecule (2a) with a
second proton (2); then applying a magnetic field (4) to the sample
and irradiating the sample with a pulse sequence (5) that
preferentially demagnetizes protons of the macromolecule (1,3)
relative to the second proton (2); allowing the second proton (2)
to exchange (6) with an exposed proton (1) of the macromolecule;
and detecting the magnetization from the relatively magnetized
second proton (2), which is now bound to the exposed position (1c)
of the macromolecule. The invention also provides a method for
observing a position in the macromolecule that bind a ligand.
Inventors: |
Sem; Daniel S.; (San Diego,
CA) ; Pellecchia; Maurizio; (San Diego, CA) |
Correspondence
Address: |
MCDERMOTT, WILL & EMERY
4370 LA JOLLA VILLAGE DRIVE, SUITE 700
SAN DIEGO
CA
92122
US
|
Assignee: |
Triad Therapeutics, Inc.
San Diego
CA
|
Family ID: |
26946760 |
Appl. No.: |
11/320157 |
Filed: |
December 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10032140 |
Dec 19, 2001 |
6979531 |
|
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11320157 |
Dec 27, 2005 |
|
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60258621 |
Dec 21, 2000 |
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60263475 |
Jan 22, 2001 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01R 33/465 20130101;
G01R 33/4608 20130101; Y10T 436/24 20150115 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method for preferentially observing an exposed position of a
macromolecule, comprising the steps of (a) obtaining a sample
comprising a macromolecule and a second molecule, wherein the
macromolecule is larger than 35 kiloDaltons and has a position that
is exposed to the second molecule, wherein a first proton is bound
to the exposed position of the macromolecule, a second proton is
bound to the second molecule, and the first proton can exchange
with the second proton; (b) applying a magnetic field to the
sample, thereby magnetizing the first proton and the second proton;
(c) irradiating the sample with a pulse sequence that
preferentially demagnetizes the protons of the macromolecule
relative to the second proton; (d) allowing the second proton to
exchange with the first proton, whereby the relatively magnetized
second proton becomes bound to the exposed position of the
macromolecule; and (e) detecting the magnetization from the second
proton; whereby the exposed position of the macromolecule is
preferentially observed.
2. The method of claim 1, wherein the macromolecule is a
polypeptide.
3. The method of claim 1, wherein the macromolecule is larger than
about 50 kDa.
4. The method of claim 1, wherein the macromolecule is larger than
about 75 kDa.
5. The method of claim 1, wherein the macromolecule is larger than
about 100 kDa.
6. The method of claim 1, wherein the structure of the polypeptide
has not been fully determined by an NMR technique.
7. The method of claim 1, wherein resonances for fewer than 5% of
the amino acids of the protein have been assigned by NMR
techniques.
8. The method of claim 1, wherein resonances for fewer than 10% of
the amino acids of the protein have been assigned by NMR
techniques.
9. The method of claim 1, wherein resonances for fewer than 50% of
the amino acids of the protein have been assigned by NMR
techniques.
10. The method of claim 1, wherein resonances for fewer than 75% of
the amino acids of the protein have been assigned by NMR
techniques.
11. The method of claim 1, wherein the second molecule is a protic
solvent.
12. The method of claim 1, wherein the second molecule is
water.
13. The method of claim 1, wherein the position on the
macromolecule that is exposed to the second molecule comprises
15N.
14. The method of claim 13, wherein the pulse sequence comprises an
.sup.15N filter.
15. The method of claim 1, wherein the pulse sequence comprises the
SEA pulse sequence.
16. The method of claim 1, wherein step (c) further comprises
.sup.15N, .sup.1H TROSY.
17. The method of claim 1, wherein the pulse sequence comprises the
SEA-TROSY pulse sequence.
18. The method of claim 1, wherein step (d) occurs during a
predetermined mixing time.
19. The method of claim 18, wherein the mixing time is between 25
and 300 ms.
20. The method of claim 18, wherein the mixing time is between 50
and 150 ms.
21-49. (canceled)
Description
[0001] This application is based on and claims benefit of U.S.
Provisional Application No. 60/258,621, filed Dec. 21, 2000, and
U.S. Provisional Application No. 60/263,475, filed Jan. 22, 2001,
both of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates broadly to the structure of
macromolecules and more specifically to nuclear magnetic resonance
(NMR) methods for preferentially observing exposed positions of a
macromolecule.
[0003] The three-dimensional structure of macromolecules such as
proteins can provide insight into their function in normal and
diseased cells. For example, when certain compounds or "ligands"
bind to a protein, the 3-D structure of the protein can help
identify the ligand's binding sites on the protein.
Atomic-resolution structures can allow researchers to understand
the interaction between ligands and macromolecules and to design
and select new ligands that may serve as therapeutic drugs.
[0004] Nuclear magnetic resonance (NMR) spectroscopy has determined
the structure of many proteins at atomic resolution. Nevertheless,
current NMR methods for protein structure determination are
extremely time-consuming because they require painstaking deduction
of the positions of thousands of individual atoms. As attention
focuses on larger and more complex proteins and the number of atoms
increases, the data generated by NMR methods can become
overwhelming, making it virtually impossible to determine the
structure of the protein.
[0005] Thus, there is a need for methods that can provide
information about large macromolecular structures in a way that is
useful for understanding ligand binding. The present invention
satisfies this need and provides related advantages as well.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provides a method for preferentially observing
an exposed position of a macromolecule. The method includes the
steps of (a) obtaining a sample including a macromolecule and a
second molecule, wherein the macromolecule is larger than 35
kiloDaltons and has a position that is exposed to the second
molecule, wherein a first proton is bound to the exposed position
of the macromolecule, a second proton is bound to the second
molecule, and the first proton can exchange with the second proton;
(b) applying a magnetic field to the sample, thereby magnetizing
the first proton and the second proton; (c) irradiating the sample
with a pulse sequence that preferentially demagnetizes the protons
of the macromolecule relative to the second proton; (d) allowing
the second proton to exchange with the first proton, whereby the
relatively magnetized second proton becomes bound to the exposed
position of the macromolecule; and (e) detecting the magnetization
from the second proton; whereby the exposed position of the
macromolecule is preferentially observed.
[0007] The invention further provides a method for observing an
exposed position in a macromolecule that binds a ligand, wherein
the macromolecule is larger than 35 kiloDaltons; has a plurality of
protons bound to positions on the macromolecule that are exposed to
the second molecule; and the exposed protons can exchange with
protons of the second molecule. The method includes the steps of
(a) performing the method set forth above with a first sample
including the macromolecule and a second molecule; (b) performing
the method set forth above with a second sample including the
macromolecule and the second molecule, wherein the macromolecule is
bound to a ligand; and (c) detecting a perturbation in the second
sample compared to the first sample; thereby observing the exposed
position in the macromolecule that binds the ligand.
[0008] A method of the invention for observing an exposed position
in a macromolecule that binds a ligand, can alternatively include a
first sample containing the macromolecule and a second molecule and
a second sample containing the macromolecule, the second molecule
and a ligand, wherein the second molecule and ligand alternatively
associate with and dissociate from the macromolecule. In another
embodiment, the method can include a first sample containing the
macromolecule, a second molecule and a first ligand, wherein the
second molecule and first ligand alternatively associate with and
dissociate from the macromolecule and a second sample containing
the macromolecule, the second molecule and a second ligand, wherein
the second molecule and second ligand alternatively associate with
and dissociate from the macromolecule. The exposed position in the
macromolecule that binds the ligand or differentially interacts
with the two ligands can be identified or characterized according
to a perturbation detected in the second sample compared to the
first sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1a to 1e schematically illustrate certain steps in the
method of the invention. The following reference numbers are used
in these Figures:
[0010] 1a macromolecule [0011] 1 first proton [0012] 1c exposed
position [0013] 3c non-exposed position [0014] 3 proton bound to
non-exposed position
[0015] 2a second molecule [0016] 2 second proton
[0017] 4 magnetic field
[0018] 5 pulse sequence
[0019] 6 exchange of protons
[0020] FIG. 2a shows a SEA-TROSY NMR pulse sequence. FIG. 2b shows
a SEA-HNCA-TROSY pulse sequence. The SEA element is enclosed by a
dashed rectangle in both figures.
[0021] FIG. 3 shows NMR spectra obtained for a catalytic portion of
cytochrome P450 reductase that has been uniformly .sup.15N/.sup.2H
labeled with protons present selectively at exchangeable positions.
A TROSY pulse sequence was used on the left side of the figure. On
the right is the corresponding spectra using the SEA-TROSY pulse
sequence with a .tau..sub.m of 100 ms.
[0022] FIGS. 4a to 4c show representative
.omega..sub.1(.sup.13C.sup..alpha.)-.omega..sub.3 (.sup.1H.sup.N)
strips from a 3D SEA-HNCA-TROSY spectrum for a 0.5 mM sample of a
.sup.15N/.sup.2H/.sup.13C labeled catalytic portion of cytochrome
P450 reductase. The assignments for the loop of residues Ala 235 to
Glu 238 (FIG. 4a), Ala 500 to Asn 503 (FIG. 4b), and Asp 632 to Ala
633 (FIG. 4c) are shown with sequential
.sup.13C.sup..alpha.(i)/.sup.13C.sup..alpha.(i-1) connectivities
indicated by the dashed line.
[0023] FIGS. 5a to 5d show various embodiments of the method of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides methods for preferentially
observing certain protons of a macromolecule 1a.
[0025] The term "macromolecule" herein means a polymeric molecule
or complex of polymeric molecules that are associated in solution,
including biological and synthetic polymers. Proteins and other
polypeptides are particularly useful biological polymers. Other
useful biological polymers include polysaccharides and
polynucleotides. Synthetic polymers include plastics and mimics of
biological polymers such as protein-nucleic acids.
[0026] The methods can be applied to relatively small
macromolecules, such as those having molecular weights of 25
kiloDaltons (kDa) or lower, and to larger proteins such as those
having molecular weights larger than 30 kDa, 35 kDa, 40 kDa or 50
kDa. The method is particularly useful when applied to very large
proteins, including those having molecular weights larger than 75
kDa, 100 kDa, 125 kDa, 150 kDa or 200 kDa. As they are used herein,
the term "molecular weight" and the particular molecular weights
described are intended to refer to the mass of a macromolecule as
it occurs in solution. Where the macromolecule occurs as a complex
of more than one molecule, the term refers to the mass of the
complex.
[0027] The methods of the invention are also useful when the
structure of a macromolecule has not been fully determined, such as
by X-ray crystallography or by an NMR technique. For example, the
method can be applied to macromolecules where resonances for fewer
than 5%, 10%, 20%, 30%, 40%, 50% or 75% of the monomer units--such
as amino acids, nucleotides or saccharide units--of the
macromolecule have been assigned by NMR. Nevertheless, when applied
to macromolecular structures that are completely or 80% to 90% to
99% determined, the method can also be useful for characterizing
the structure and distinguishing between the functions of
individual atoms.
[0028] Sources for macromolecules can be natural, recombinant or
synthetic. For example, they can be obtained from biological cells
or tissues that naturally produce the macromolecule, or from
recombinant cells that have been modified to express the
macromolecule.
[0029] Natural macromolecules can be isolated from various sources
by methods known in the art. For example, polynucleotides can be
isolated according to methods described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 3.sup.rd Ed., Cold Spring
Harbor Press, Plainview, N.Y. (2001) and Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY
(1992), and in Ausebel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (2000). Methods for
isolating polypeptides and proteins from cells and tissues are
described in Scopes, Protein Purification: Principles and Practice,
3d ed., Springer-Verlag, New York N.Y. (1994); Duetscher, Methods
in Enzymology, vol. 182, Academic Press, San Diego Calif. (1990);
and Coligan et al., Current Protocols in Protein Science, John
Wiley and Sons, Baltimore Md. (2000). Recombinant expression
systems and methods are also described in Goeddel, Methods in
Enzymology, vol. 185, Academic Press, San Diego Calif. (1990); Wu,
Methods in Enzymology, vol. 217, Academic Press, San Diego Calif.
(1993); Sambrook et al., supra (2001), and Ausebel et al., supra.
(2000).
[0030] Polymeric macromolecules can be produced by well known
synthetic methods. A polypeptide or protein can be produced by
synthetic methods including Merrifield solid-phase synthesis,
t-Boc-based synthesis, Fmoc synthesis and variations on these
techniques. Polynucleotides and oligonucleotides can be produced
using well known synthetic methods such as those incorporating
phosphoramidite chemistry.
[0031] Particularly useful macromolecules can be isotopically
labeled with .sup.2H, .sup.13C, .sup.15N or any combination of
these isotopes. For example, a protein or polypeptide can be
isotopically labeled by adding metabolites such as amino acids to
the culture medium of a cell or tissue producing the protein or
polypeptide as described in Venters et al., J. Biomol. NMR
5:339-344 (1995). Isotope labels can be incorporated into synthetic
macromolecules using isotopically labeled reactants under otherwise
standard conditions of synthesis.
[0032] The term "proton of a macromolecule" or "first proton" 1
used herein means a proton bound to another atom of the
macromolecule. The term "bound" used herein refers to when two
atoms interact directly, whether ionically, covalently or via
hydrogen bonding. The protons can be bound to any atom of the
macromolecule, but are typically bound to atoms having
electronegative charge or polarity including an oxygen, nitrogen or
sulfur. The interaction can be stable or transient so long as it is
maintained for sufficient time to detect magnetic coupling between
the nuclei. Thus, a proton bound to a macromolecule need not be
permanently bound, but can be dissociable. Protons can be
dissociable when they exchange with solvent on a timeframe of
hours, minutes, seconds, microseconds or microseconds or
faster.
[0033] The term "second molecule" 2a used herein means any molecule
that is not the macromolecule, but has at least one bound proton 2
that is capable of being exchanged. The bound proton of the second
molecule is designated as the "second proton" to distinguish it
from the first proton, which is bound to the macromolecule.
[0034] A useful second molecule is a solvent molecule. The term
"solvent" or bulk solvent used herein means molecules that solvate
a macromolecule and interact transiently with a surface of the
macromolecule. The term "protic solvent" used herein means a
solvent that has a proton that is bound to the rest of the solvent
molecule or is solvated by a component of the sample other than a
macromolecule. A protic solvent can contain a polar X--H bond where
X is a heteroatom with a lone pair such as N, O or S. Typically the
solvent surrounds the surface of the macromolecule, but can also
interact with subregions of the macromolecule, such as when a
membrane protein is fully or partially within a lipid bilayer.
[0035] It follows that the term "exposed" used herein means a
proton or position on the macromolecule that can interact directly
with the second molecule. An exposed position of a macromolecule
can be one or more atoms on the exterior surface of a macromolecule
or within an internal surface such as a channel, cavity or pocket
so long as the position can be in direct contact with the second
molecule. Furthermore, when a macromolecule can exist as a
multimeric complex, individual submits may have positions that are
exposed when monomeric, but relatively less exposed or not exposed
when complexed. In contrast, a non-exposed atom 3 is restricted
from contacting the second molecule, for example due to steric
constraints imposed by the macromolecule, shared bonding between
two atoms as in a hydrogen bond or location within a hydrophobic
region of the macromolecule. Thus, even if the second molecule is
not in immediate contact, but can be moved toward and interact with
the position on the macromolecule, then the position is exposed;
interaction need not be constant or permanent.
[0036] Exposed positions on the macromolecule are exposed to bulk
solvent. Such solvent-exposed positions can be particularly useful
in the methods of the invention because ligands are typically
brought into contact with solvent exposed positions of
macromolecules. Thus, solvent-exposed positions are likely
candidates for binding-positions of a macromolecule, where natural
ligand or nonnatural ligand mimics can bind. It follows that
solvent-exposed positions of a macromolecule can be of particular
interest to researchers of ligand-macromolecule interactions.
[0037] This invention provides NMR methods for preferentially
observing positions of macromolecules that are in such exposed
positions. When previous NMR methods were applied to large
macromolecules, they detected resonances corresponding to
potentially every atom in the macromolecule. The resulting spectra
have been overwhelmingly complex so that the sheer number of
overlapping resonances have required months, even years to
interpret. In many cases, full interpretation is impossible.
Ironically, the majority of the atoms are of little interest to
researchers trying to understand the interaction between
macromolecules and ligands. By focusing on exposed positions, the
present invention enables researches to concentrate on likely
ligand-binding positions on macromolecules.
[0038] As illustrated in FIG. 1a, the method of the invention
begins with a sample having a macromolecule 1a and a second
molecule 2a. The macromolecule has a proton or "first proton" 1
bound to a position 1c that is exposed to the second molecule. The
macromolecule has other protons 3 as well, bound to non-exposed
positions 3c. The second molecule has a proton or "second proton" 2
that can exchange with the first proton 1.
[0039] In FIG. 1b, a magnetic field 4 is applied to magnetize the
protons in the sample, including the first proton 1, the second
proton 2, as well as non-exposed protons 3. As illustrated in FIG.
1c, the sample is then irradiated with a pulse sequence 5 that
preferentially demagnetizes protons of the macromolecule 1,3
compared to the second proton 2. A duration of time called the
mixing time or .tau..sub.m, is allowed to pass during which the
second proton 2, which has been relatively magnetized, exchanges 6
with the first proton 1, as shown in FIG. 1d.
[0040] The term "exchange" when applied herein to first and second
protons means the first proton is replaced by a proton of the
second molecule. Thus, the first proton dissociates from an exposed
position of a macromolecule and associates with a second molecule;
likewise, the second proton dissociates from a second molecule and
associates with the macromolecule. Thus, the term "exchanged
proton" means a proton that was previously associated with one
molecule, but is now associated with a different molecule.
[0041] As a result of the proton exchange, the second proton 2
becomes bound to the macromolecule at the exposed position 1c.
Because the second proton is relatively magnetized and is now bound
to the exposed position of the macromolecule 1c, the exposed
position can be preferentially observed using NMR methods. By
comparison, protons of the macromolecule that are not exposed 3
will be relatively less magnetized due to the demagnetizing pulse
sequence, and will be less detectable by the NMR methods. As shown
in FIG. 1e, the result is the ability to preferentially observe
positions of a macromolecule that are in solvent-exposed positions
1c.
[0042] Rather than detect complex resonances of all atoms in a
large macromolecule, the resonances arising from atoms outside of
the exposed regions can be removed, thereby minimizing overlap of
peaks in a spectrum. As a result, the invention provides increased
discrimination of resonances from regions of the macromolecule that
are of particular interest for understanding ligand binding.
NMR Methods
[0043] These NMR methods use the phenomenon of nuclear magnetic
resonance, which occurs when a nucleus aligned with an applied
magnetic field is induced to absorb energy and change spin
orientation with respect to the applied field. The energy at which
this orientation change occurs is the resonance energy for the
nucleus.
[0044] The term "resonance" used herein means a signal arising when
an atom in a magnetic field absorbs radiofrequency radiation. Such
a signal can be indicated as a peak or other component of a
spectrum measured for a macromolecule in an NMR experiment. For a
particular atom, the resonance energy is a function of physical
properties of the atom and its nucleus, such as the ratio of its
magnetic moment to angular momentum or magnetogyric ratio, and the
strength of the magnetic field experienced by the nucleus. Thus, an
unshielded nucleus in an applied magnetic field will have a
resonance energy at the Larmor frequency, which is defined by the
strength of the field and its magnetogyric ratio. Generally, atoms
in a molecule have their nuclei shielded from the applied magnetic
field due to surrounding electrons from other atoms in the
molecule. As a result, the shielded nucleus will experience a local
magnetic environment that is different from the applied field, and
its resonance will be shifted from its Larmor frequency.
[0045] NMR spectroscopy can be used to obtain a spectrum of signals
arising from individual atoms of a molecule. The shift from the
Larmor frequency can generally be used to determine the relative
location of individual atoms in a molecule. These relative
locations can be used to determine the structure of the molecule.
However, large molecules, such as macromolecules, contain many
atoms, all having resonances within a small spectral region around
the Larmor frequency. Such macromolecules can produce complex
spectra with overlapping signals that are difficult to resolve.
Therefore, limiting detection to a subset of these atoms can
provide advantages for interpreting spectra of large
macromolecules.
[0046] The term "assigned" used herein in reference to a resonance
measured by NMR means the identification of a location for one or
more atoms in the sequence or structure of a macromolecule
corresponding to this resonance. One or more atom can be located in
a structure of a macromolecule relative to a structure for a
portion of the macromolecule such as a structure of an amino acid
or portion of the structure.
[0047] Any atomic nucleus that has either odd mass or odd atomic
number, or both, has a magnetic moment and can be detected by NMR
methods, including .sup.1H, .sup.13C, .sup.15N, .sup.19F and
.sup.31P. The methods of the invention can be used to
preferentially observe any NMR detectable atom that exchanges a
demagnetized proton with a magnetized proton from the second
molecule, such as a solvent. For biological macromolecules in
solvents having pH in the physiological range, an NMR detectable
atom that can exchange with a second proton is .sup.15N. A protein
can contain .sup.15N atoms with exchangeable protons at a number of
locations including at an amino terminus, backbone amide, lysine
e-amine, histidine imidizole, arginine guanidinium, tryptophan or
asparagine or glutamine amide.
[0048] The term "magnetized" used herein means a state of the
nucleus where it has a magnetic moment aligned with respect to a
magnetic field. The term is intended to include an atomic nucleus
in an applied magnetic field having a magnetic moment aligned with
or against the direction of the applied magnetic field. A
population of atomic nuclei in an applied magnetic field will have
members aligned both with and against the direction of the magnetic
field.
[0049] A magnetic field that magnetizes protons in the methods of
the invention can be of any strength sufficient to cause the
magnetic dipole of the proton to align with the field. Magnetic
field strengths useful in the methods of the invention can be
relatively low including 60 to 100 MHz. The methods can also
exploit higher resolution available at higher field strengths
including at least about 400 MHz, 500 MHz, 600 MHz, 750 MHz, 800
MHz, 900 MHz or higher. Those skilled in the art will know or be
able to determine how to produce a polarizing magnetic field using
methods known in the art as described, for example, in Fukushima
and Roeder, Experimental Pulse NMR Adams and Wesley (1981).
[0050] A sample used in the methods of the invention can be
irradiated with a variety of pulse sequences 5 that preferentially
demagnetize a proton bound to an atom of a macromolecule compared
to exchangeable second protons.
[0051] The term "pulse sequence" used herein means a temporal
series of one or more radiofrequency irradiations of defined
duration, intensity, frequency and/or relative polarization with
respect to a magnetic field and/or one or more magnetic field
gradients of defined duration, field strength and relative
orientation.
[0052] The term "demagnetize" used herein means the process or
effect of irradiating the atomic nucleus so that net absorption or
emission of energy from a second irradiation is reduced or
undetectable. The term is intended to include irradiation of any
frequency, duration, intensity or polarization that causes a signal
from an atomic nucleus to be reduced or removed from an NMR
spectrum. The term includes radio frequency radiation, including
oscillating electrical voltages, currents or electromagnetic fields
with frequencies in the range of 10 to 1000 MHz or 10.sup.7 to
10.sup.9 sec.sup.-1. The frequency selected depends on the magnetic
field strength and the corresponding Larmor frequency for the
nucleus of interest at that field strength.
[0053] An example of a pulse sequence that can preferentially
demagnetize a proton bound to a nitrogen compared to a water proton
is the .sup.15N double filter of the SEA pulse sequence described
in Example I and illustrated in FIG. 2. One skilled in the art will
recognize that modifications can be made in the .sup.15N double
filter of the SEA pulse sequence as long as the pulse sequence
preferentially demagnetizes a proton bound to a nitrogen compared
to a water proton. Modifications can include the duration or
strength of pulsed field gradients; the intensity, phase, duration
or sequence of the pulses; or the duration of the delay .tau.
between pulses. In addition, other pulse sequences that
preferentially demagnetize a proton bound to a nitrogen compared to
a water proton can be used in the SEA pulse sequence including the
.sup.15N filters described in Breeze, Prog. NMR Spectr. 36:323-372
(2000) and Otting and Wuthrich, Quarterly Rev. Biophys. 23:39-96
(1990).
[0054] Signals arising from a sample irradiated with the
demagnetizing pulse sequence are detected at the end of the mixing
time. The term "mixing time" or .tau..sub.m is used herein to mean
a time period long enough for a dissociable proton bound to a
position of a molecule to exchange with a second proton, but
insufficient for a dissociable proton bound to a second position of
the molecule to exchange with a second proton. For example, the
term can refer to a time period long enough for a dissociable
proton bound to a position of a macromolecule to exchange with a
solvent proton, but insufficient for a dissociable proton bound to
a second position of the macromolecule to exchange with a solvent
proton. The term can include a time period during which second
protons preferentially exchange with an exposed proton of a
macromolecule compared to a buried proton of the macromolecule. The
term can also include a time period resulting in preferential
exchange of a magnetized second proton with an exposed proton bound
to an atom of a macromolecule compared to exchange of a magnetized
second proton with a buried proton bound to the macromolecule.
[0055] The mixing time can be any time period sufficient for a
demagnetized proton bound to an exposed position of a molecule to
exchange with a magnetized second proton and insufficient for a
demagnetized proton, bound to an atom in the interior of the same
molecule to exchange with a magnetized second proton. It follows
that measurement of signals arising from a macromolecule at
.tau..sub.m will preferentially detect exposed positions that have
acquired a magnetized proton from the second molecule, while atoms
of the macromolecule that are buried in the interior of the
macromolecule will not be detected.
[0056] A mixing time that provides preferential detection of
exposed positions can be in the range of about 25 to 500 msec. The
number of signals that can be detected following a demagnetizing
pulse sequence of the invention is proportional to the mixing time,
so that shorter mixing times will result in fewer atoms of a
macromolecule acquiring a magnetized second proton, thereby
yielding a smaller number of detected signals. Thus, the invention
can be used with shorter mixing times in the range of about 25 msec
up to about 50, 100 or 150 msec. A similar relationship exists
between the intensity of a detected signal and mixing time, so that
increased mixing time will allow more of the macromolecules in a
sample to acquire a magnetized second proton at a particular atom,
thereby increasing signal for that particular atom. Accordingly,
the invention can be used with longer mixing times in the range of
about 150 msec up to about 200, 300, 400 or 500 msec.
[0057] The methods of the invention can be performed in an
iterative fashion to determine a mixing time to achieve sufficient
signal detection characteristics, including signal resolution,
signal intensity or number of signals in a spectrum. Specifically,
the methods can be performed using an initial .tau..sub.m in the
range described above and the resulting signal or spectrum can be
evaluated for a desired signal detection characteristic. The
methods can then be iteratively repeated with systematic changes in
.tau..sub.m until a sufficient or desired characteristic is
achieved.
[0058] Additionally, a mixing time can be determined based on a
theoretically estimated-proton exchange rate or an empirically
measured rate for proton exchange in a macromolecule of interest.
An appropriate .tau..sub.m can be determined from a proton exchange
rate constant k.sub.ex according to the following relationship:
I=I.sub.m(1-e.sup.-kex*tm) where I is the intensity of the signal
detected and I.sub..infin. is the intensity at infinite mixing
time. NMR-based methods for determining proton exchange rates for
atoms with previously assigned resonances are known and described
in Gemmecker et al., J. Am. Chem. Soc. 115:11620-11621 (1993) and
Bai et al., Prot. Struct. Func. Gen. 17: 75-86 (1993). A proton
exchange rate or proton exchange rate constant determined by such
methods can be used to determine an appropriate .tau..sub.m for
selectively observing a subset of atoms in the macromolecule, such
as a subset including those atoms.
[0059] Magnetization from an exchanged proton bound to an atom of a
macromolecule can be detected using methods known in the art of NMR
spectroscopy. Any method for determining dipolar or scalar coupling
between a magnetized proton and a bound or proximal atom can be
used in the invention. Thus, the methods can be performed in
combination with a variety of pulse sequences or heteronuclear
filters. One skilled in the art will know or be able to determine
an appropriate pulse sequence or filter for use in the methods of
the invention including those described in Cavanaugh et al.,
Protein NMR Spectroscopy: Principles and Practice, Academic Press,
San Diego Calif. (1996).
[0060] A further advantage of the invention is that the methods can
be used in combination with a variety of high resolution NMR
techniques known in the art to increase the range of macromolecules
suitable for structure analysis.
[0061] The method of the invention can further incorporate the step
of determining a heteronuclear correlation measurement for the
sample, where one of the correlated nuclei is .sup.1H, such as a
.sup.15N--.sup.1H correlation measurement. Any method for
determining a signal or spectrum arising from an .sup.15--.sup.1H
correlation can be used in the invention including two-dimensional
methods such as COSY, NOESY or TROSY techniques. These methods can
employ filters to select for particular resonance signals such as
an .sup.15N filter which can be used in a [.sup.1H--.sup.1H] NOESY
to select for .sup.15N attached protons as described in Cavanaugh
et al., supra (1996). Pulse sequences for these .sup.15--.sup.1H
correlation techniques can be used in the methods of the invention
as described further below.
[0062] COSY refers to Correlation Spectroscopy and includes methods
that produce signals arising from coupling between magnetized
hydrogen atoms that are covalently connected through one or two
other atoms. COSY pulse sequences and methods are known in the art
as described in Cavanaugh et al., supra (1996). One skilled in the
art will be able to perform the methods of the invention in
combination with a COSY pulse sequence by combining the COSY pulse
sequence with a pulse sequence of the invention according to the
methods exemplified in Example I for combining a SEA pulse sequence
with a TROSY pulse sequence.. The methods of the invention can be
advantageously used in combination with a COSY technique to reduce
the number of peaks present in a two-dimensional COSY spectrum of a
macromolecule, thereby providing improved resolution and enhanced
assignment of structure or function of the macromolecule.
[0063] TROSY refers to Transverse Relaxation Optimized Spectroscopy
which is a method that reduces line broadening, due to transverse
relaxation (T.sub.2), in a spectrum of a molecule having two
coupled spin 1/2 nuclei, by using chemical shift anisotropy to
cancel out relaxation effects due to cross correlation between
dipolar coupling of the two spin 1/2 nuclei. TROSY provides a
.sup.15--.sup.1H correlation technique that minimizes signal
overlap compared to a standard correlation spectrum.
[0064] A combination of TROSY with the methods of the invention can
provide particular advantages due to the synergistic effects of
combining reduced peak broadening in a spectrum achieved with TROSY
and reduction in the number of signals present in a spectrum
achieved with the methods of the invention. Specifically, this
synergism can simplify a spectrum of a large macromolecule to
provide an improved resolution compared to the use of either method
in isolation. The TROSY technique and associated pulse sequences
are described in U.S. Pat. No. 6,133,736; Wider and Wuthrich,
Current Opinion in Structural Biology 9:594-601 (1999); and
Pervushin et al., Proc. Natl. Acad. Sci. USA 94:12366-12371 (1997).
One skilled in the art will know that the TROSY pulse sequence can
include variants of the pulse sequences described and will be able
to recognize a TROSY pulse sequence accordingly.
[0065] NOESY or Nuclear Overhauser Effect Spectroscopy refers to a
method that can be used to determine, through space, the proximity
of atoms in a molecule according to an interaction between proximal
nuclei, called the Nuclear Overhauser Effect, that results in
transfer of magnetization between the proximal nuclei. A
two-dimensional NOESY spectrum contains crosspeaks for protons that
are sufficiently proximal to have an NOE interaction. Techniques
and pulse sequences for determining NOESY spectra are known in the
art, as described in Cavanaugh et al., supra (1996). Since an NOE
occurs by spatial proximity, not merely connection via chemical
bonds, it is especially useful for determining distances between
molecules. The methods of the invention are especially useful for
simplifying spectra produced by NOESY techniques and provide the
advantage of improving identification of signals arising from
proximal nuclei in a macromolecule or between a macromolecule and
ligand that would otherwise be difficult or impossible to resolve
in the NOESY spectrum alone.
[0066] The invention also provides a method for determining an NMR
correlation between three nuclei. The method further involves
determining a second correlation measurement between the correlated
nuclei and a third nucleus.
[0067] In one embodiment of the invention, selective observation of
a subset of atoms in a molecule can be used with multidimensional
NMR techniques. Multidimensional NMR techniques are known in the
art and can be used to measure correlation between a number of
different heteronuclei equivalent to the number of dimensions
included. For example, a three-dimensional NMR spectrum can be used
to determine a heteronuclear NMR correlation between three
different heteronuclei such as .sup.1H, .sup.15N and .sup.13C.
[0068] As with spectra produced from one- and two-dimensional
techniques, a three-dimensional spectrum of a macromolecule can be
complicated due to the large number of detected signals that can
often overlap, resulting in poor resolution of signals. Selective
observation of a subset of atoms in a molecule can simplify a
three-dimensional spectrum by reducing signal overlap. For example,
selective observation of exposed positions in a macromolecule using
a 3-D NMR technique can resolve peaks in a three-dimensional
spectrum that arise from exposed atoms by removing overlapping
signals arising from buried atoms.
[0069] The methods of the invention can include determination of
correlation between an exchanged magnetized proton, an atom to
which it is bound and any third, magnetically active heteronucleus
including .sup.13C. Also included is the selection of either an
.sup.15N or .sup.1H followed by correlation with two other atoms. A
variety of well known triple resonance methods can be used to
determine correlation between an exchanged magnetized proton, an
atom to which it is bound and .sup.13C. Such methods include HNCA,
HNCACB, CBCA(CO)NH and HNCO. These methods are described in
Cavanaugh et al., supra (1996) and share the common feature of
having .sup.1H and .sup.15N dimensions that can be selected with
the SEA element. The third dimension is frequently .sup.13C, and of
different atom types such as alpha carbons (C.sub..alpha. or CA) or
carbonyl carbons (CO). Another triple resonance experiment that can
be simplified using the methods of the invention is .sup.15N edited
NOESY.
[0070] An example of selective observation of exposed atoms in a
macromolecule is provided in Example 2, which describes assignment
of NMR signals to specific regions in a large protein structure
using spectra obtained with a 3D SEA-HNCA-TROSY pulse sequence. A
3D SEA-HNCA-TROSY spectrum can be particularly useful for
determining backbone resonance assignments for exposed loop regions
of a protein, as demonstrated in Example II.
[0071] The methods of the invention can therefore be used to
selectively detect atoms in any macromolecule based on differences
in the proton exchange rates. Specifically, the methods can be used
to preferentially detect the subset of atoms in positions that have
exchanged a proton with a second proton in a defined time period
compared to another atom in the molecule that has not exchanged a
bound proton.
[0072] Therefore, the methods can be used to selectively detect an
atom or subset of atoms according to factors such as steric
accessability to solvent or another second molecule, thermodynamic
stability of the atom-hydrogen bond, pKa or chemical environment of
the atom. A subset of atoms of a molecule that can be
preferentially detected according to steric accessability to second
molecules include positions on the surface of a molecule that can
be preferentially detected compared to positions in the interior of
the same molecule. A particular subset of atoms includes those that
exchange on the 1-1000 msec timescale, relative to others that are
more stable such as carbon attached protons. The methods can also
be used to detect atoms having more stable proton bonds than other
atom-proton bonds, caused by hydrogen bond interactions with a
second atom or differences in local polarity, hydrophobicity or
hydrophilicity. In particular, the amide nitrogens present in a
protein can have different stabilities due to their occurrence in a
variety of local protein environments and ability to participate in
hydrogen bond interactions in alpha-helices or beta-sheet
structures. Thus, preferential observation of amide signals by the
methods of the invention can be useful for structural and
functional characterization of proteins.
[0073] The invention also provides detection according to extrinsic
factors influencing the atoms of the molecule. Specifically,
different atoms or subsets of atoms can be preferentially detected
by performing the methods of the invention under different
conditions such as pH, temperature, polarity or presence of a
ligand.
[0074] The methods can be used in combination with changes in
extrinsic factors to select a different atom or subset of atoms
that is preferentially detected. For example, by changing the pH of
a sample, different subsets of atoms can be detected according to
the differences in pKa for atoms in the molecule. The resulting
combination of spectra can provide a larger number of resolved
signals when compared to a single spectrum, thereby increasing the
number of resolved signals that can be used for structure
determination of the molecule. Additionally, comparison of signals
or subsets of signals that result under different conditions can be
used to determine dynamics or function of the molecule.
Ligand Methods
[0075] The use of ligands expands the possible applications for the
method of the invention. For example, the second molecule can be a
ligand, such as a ligand in a bulk solvent (FIG. 5a and 5c) or
present in a lipid bilayer (FIG. 5c). The macromolecule in the
sample can also be bound to a ligand 9, whether at an exposed
position (FIG. 5d) or a non-exposed position.
[0076] The invention further provides a method for identifying an
exposed position in a macromolecule that binds a ligand, where the
macromolecule has a plurality of protons bound to positions on the
macromolecule that are exposed to the second molecule, and the
exposed protons can exchange with protons of the second molecule.
The method involves the steps of performing the method described
above with samples of macromolecule bound (FIG. 5b) and not bound
(FIG. 5a) to a ligand 8. Then chemical shift perturbations are
detected in the bound sample compared to the unbound sample. In
addition to chemical shift perturbation, other changes that can be
measured include reduced signal intensity or differential proton
exchange between the samples. As a result, the method identifies
the exposed position in a macromolecule that binds the ligand.
[0077] The ligand binding sites of macromolecules generally contain
atoms that are bound to rapidly exchanging protons and are often
located in exposed regions, whereas atoms buried in the interior of
a macromolecule or involved in hydrogen bonding are unlikely to
interact with a ligand. For example, loop regions of proteins are
often primary components of protein binding site regions. A
majority of atoms located in protein loop regions are typically not
involved in hydrogen bonds or secondary structure interactions and
are thus prone to rapid exchange with second protons. Therefore,
selective observation of exposed atoms in a protein using the
methods of the invention can provide selective observation of those
atoms that interact with a ligand.
[0078] The methods of the invention can be used to observe an
exposed position in a macromolecule that binds a ligand where the
macromolecule has a plurality of protons bound to positions on the
macromolecule that are exposed to the second molecule, and the
exposed protons can exchange with protons of the second molecule.
The method involves the steps of performing the method described
above with samples of macromolecule in the presence and absence of
a ligand, wherein the second molecule and ligand alternatively
associate with and dissociate from the macromolecule. The exposed
position can be identified or characterized according to a
perturbation in the presence of the ligand compared to in the
absence of the ligand. Similarly, the method can involve the steps
of performing the method with a first sample of a macromolecule in
the presence of a first ligand and a second sample of a
macromolecule in the presence of a second ligand, wherein each of
the ligands can associate with and dissociate from the
macromolecule alternatively with the second molecule. The exposed
position can be identified or characterized according to a
perturbation in the presence of the first ligand compared to in the
presence of the second ligand.
[0079] As used herein, the term "perturbation" refers to the act of
changing or state of being changed. The term can include an NMR
perturbation which is the act of magnetizing or demagnetizing an
atomic nucleus or the resulting magnetization state of a nucleus.
The term can also include a sample perturbation, which is the act
of changing one sample compared to another or the state of one such
sample compared to the other, in separate, but substantially
similar experiments. Resulting changes occurring in such samples
can be observed as detected perturbations and include significant
difference in an NMR measurement or spectrum for one of the samples
compared to the other.
[0080] The invention provides advantages for determination of
macromolecule-ligand interactions in that the methods can produce a
spectrum where signals arising from slower exchanging atoms, which
tend not to interact with a ligand, can be removed to minimize
potential overlap with signals arising from atoms located in a
ligand binding site. Thus, the methods allow high resolution
characterization of protein-ligand interactions for larger
macromolecules than would be possible without selective observation
of binding site atoms.
[0081] The invention can be used with any ligand that interacts
with a macromolecule with sufficient stability to be observed in
the time frame of an NMR experiment, including chemical or
biological molecules such as simple or complex organic molecules,
metal-containing compounds, carbohydrates peptides,
peptidomimetics, carbohydrates, lipids and nucleic acids. Thus, an
interaction between proteins or subunits of a protein can be
considered as a macromolecule-ligand binding interaction where one
of the proteins or subunits is considered a ligand for the other
protein or subunit. The methods can be used to observe interactions
of ligands having affinities for a macromolecule in a variety of
ranges observable by NMR methods. The composition of the sample can
be manipulated to alter the rates of association or dissociation of
a ligand with a macromolecule. Factors that can be manipulated
include the type of ligand used, pH of the sample, concentration of
the ligand or macromolecule or temperature. Using such
manipulations those skilled in the art can provide conditions where
the rate at which the ligand associates with the macromolecule is
slower than or comparable to the rate at which the exposed protons
of the macromolecule exchange with protons of the second molecule.
Thus, conditions can be provided where the rate at which the ligand
associates with the macromolecule is at most 10, 100 or 1000 fold
higher than the rate at which the exposed protons of the
macromolecule exchange with protons of the second molecule. As set
forth above, the time allowed for proton exchange to occur can also
be adjusted to suit a particular ligand and its dissociation and
association rates.
[0082] The term "ligand" used herein means a molecule that can
specifically bind to a macromolecule. "Specific binding" means that
the binding is detectable over non-specific interactions by
quantifiable assays well known in the art. A ligand can be
essentially any type of natural or synthetic molecule including a
protein, polypeptide, nucleic acid, carbohydrate, lipid, amino
acid, nucleotide or any organic-derived compound. The term also
encompasses a cofactor or a substrate of a polypeptide having
enzymatic activity or substrate that is inert to catalytic
conversion by the bound polypeptide.
[0083] In one embodiment, the methods of the invention can be used
with a ligand that is a nucleotide derivative, including a
nicotinamide adenine dinucleotide-related molecule. Nicotinamide
adenine dinucleotide-related (NAD-related) molecules that can be
used in the methods of the invention include oxidized nicotinamide
adenine dinucleotide (NAD.sup.+), reduced nicotinamide adenine
dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide
phosphate (NADP.sup.+) and reduced nicotinamide adenine
dinucleotide phosphate (NADPH).
[0084] Although ligands that naturally bind to the macromolecule
can be used, ligand mimics can also be used in place of a natural
ligand. A mimic is a molecule that has at least one function that
is substantially the same as a function of a second molecule. A
mimic of a ligand can be identified according to its ability to
bind to the same sites on a macromolecule as the ligand. For
example, a mimic can be identified by a binding competition assay
using a ligand and a mimic. The structure of a mimic can be similar
or different compared to the structure of the ligand it mimics as
long as the two molecules bind to the same binding site region on a
macromolecule. The term can encompass molecules having portions
similar to corresponding portions of the ligand in terms of
structure or function.
[0085] Examples of mimics to the ligand NADH, including Cibacron
Blue, are described in Dye-Ligand Chromatography, Amicon Corp.,
Lexington. Mass. (1980). Numerous other examples of NADH-mimics,
including useful modifications to obtain such mimics, are described
in Everse et al., The Pyridine Nucleotide Coenzymes, Academic
Press, New York N.Y. (1982). Particular analogs include
nicotinamide 2-aminopurine dinucleotide, nicotinamide
8-azidoadenine dinucleotide, nicotinamide 1-deazapurine
dinucleotide, 3-aminopyridine adenine dinucleotide, 3-acetyl
pyridine adenine dinucleotide, thiazole amide adenine dinucleotide,
3-diazoacetylpyridine adenine dinucleotide and 5-aminonicotinamide
adenine dinucleotide. Particular mimics can be identified and
selected by ligand-displacement assays, for example using
competitive binding assays with a known ligand. Mimic candidates
can also be identified by searching databases of compounds for
structural similarity with the ligand or a mimic.
[0086] In another embodiment, the methods of the invention can be
used with a ligand that is an adenosine phosphate-related molecule.
Adenosine phosphate-related molecules can be selected from the
group consisting of adenosine triphosphate (ATP), adenosine
diphosphate (ADP), adenosine monophosphate (AMP) and cyclic
adenosine monophosphate (cAMP). An adenosine phosphate-related
molecule can also be a mimic of these molecules. Mimics of an
adenosine phosphate-related molecule include quercetin,
adenylylimidodiphosphate (AMP-PNP) or olomoucine.
[0087] A ligand useful in the methods of the invention can be a
cofactor, coenzyme or vitamin including NAD, NADP or ATP as
described above. Other examples include thiamine (vitamin B.sub.1),
riboflavin (vitamin B.sub.2), pyridoximine (vitamin B.sub.6),
cobalamin (vitamin B.sub.12), pyrophosphate, flavin adenine
dinucleotide (FAD), flavin mononucleotide (FMN), pyridoxal
phosphate, coenzyme A, ascorbate (vitamin C), niacin, biotin, heme,
porphyrin, folate, tetrahydrofolate, nucleotide such as guanosine
triphosphate, cytidine triphosphate, thymidine triphosphate,
uridine triphosphate, retinol (vitamin A), calciferol (vitamin
D.sub.2), ubiquinone, ubiquitin, a-tocopherol (vitamin E),
farnesyl, geranylgeranyl, pterin, pteridine and S-adenosyl
methionine (SAM).
[0088] A protein or polypeptide can be used as a ligand in the
invention. Examples of protein and polypeptide ligands include
naturally occurring polypeptide ligands such as ubiquitin or
polypeptide hormones including insulin, human growth hormone,
thyrotropin releasing hormone, adrenocorticotropic hormone,
parathyroid hormone, follicle stimulating hormone, thyroid
stimulating hormone, luteinizing hormone, human chorionic
gonadotropin, epidermal growth factor and nerve growth factor. In
addition a ligand useful in the methods of the invention can be a
non-naturally occurring protein or polypeptide that has binding
activity. Such ligands can be identified by screening a synthetic
polypeptide library, such as a phage display library or
combinatorial polypeptide library as described below. A protein or
polypeptide ligand can also contain amino acid analogs or
derivatives such as those described below. Methods of isolation of
protein or polypeptide ligands are well known in the art and are
described in Scopes, supra; Duetscher, supra; and Coligan et al.,
Current Protocols in Protein Science, John Wiley and Sons,
Baltimore, Md. (2000).
[0089] A nucleic acid can also be used as a ligand in the
invention. Examples of nucleic acid ligands useful in the invention
include DNA, such as genomic DNA or cDNA or RNA such as mRNA,
ribosomal RNA or tRNA. A nucleic acid ligand can also be a
synthetic oligonucleotide. Such ligands can be identified by
screening a random oligonucleotide library for ligand binding
activity as described below. Nucleic acid ligands can also be
isolated from a natural source or produced in a recombinant system
using well known methods in the art including those described in
Sambrook et al., supra; Ausubel et al., supra.
[0090] A ligand used in the invention can be an amino acid, amino
acid analog or derivatized amino acid. An amino acid ligand can be
one of the 20 essential amino acids or any other amino acid
isolated from a natural source. Amino acid analogs useful in the
invention include neurotransmitters, such as .gamma.-amino butyric
acid, serotonin, dopamine, or norepinephrine or hormones such as
thyroxine, epinephrine or melatonin. A synthetic amino acid or
analog can also be used in the invention. A synthetic amino acid
can include chemical modifications of an amino acid such as
alkylation, acylation, carbamylation, iodination or any
modification that derivatizes the amino acid. Such derivatized
molecules include those molecules in which free amino groups have
been derivatized to form amine hydrochlorides, p-toluene sulfonyl
groups, carbobenzoxyl groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups can be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups can be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of
histidine can be derivatized to form N-benzylhistidine. Naturally
occurring amino acid derivatives of the twenty standard amino acids
can also be included in a cluster of bound conformations including
4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,
ornithine and carboxylglutamate.
[0091] A lipid ligand can also be used in the invention. Examples
of lipid ligands include triglycerides, phospholipids, glycolipids
or steroids. Steroids useful in the invention include
glucocorticoids, mineralocorticoids, androgens, estrogens and
progestins.
[0092] Another type of ligand that can be used in the invention is
a carbohydrate. A carbohydrate ligand can be a monosaccharide such
as glucose, fructose, ribose, glyceraldehyde and erythrose; a
disaccharide such as lactose, sucrose or maltose; oligosaccharide
such as those recognized by lectins such as agglutinin, peanut
lectin or phytohemagglutinin, or a polysaccharide such as
cellulose, chitin or glycogen.
[0093] An atom in a macromolecule that binds a ligand can be
identified with the methods of the invention according to
differential exchange of protons at the atom in the presence and
absence of the ligand. Specifically, an atom in a macromolecule can
be identified as interacting with the ligand when it is protected
from acquiring a magnetized proton from the second molecule when
the ligand is present to a greater extent than when the ligand is
absent. For example, SEA-TROSY NMR spectra acquired for a
macromolecule in the presence and absence of a ligand can be
compared. NMR cross peaks that change intensity or chemical shift
between the two spectra can be identified as corresponding to atoms
that interact with the ligand. The improved signal resolution and
spectral simplification resulting from selective observation of the
subset of atoms involved in ligand binding can provide improved
detection of changes in intensity or chemical shift for large
macromolecules due to removal of potentially overlapping signals
arising from atoms not involved in ligand binding.
[0094] The methods can also be used to determine the structure and
dynamics of a ligand binding site. Three-dimensional NMR techniques
such as those described above can be used in combination with
selective observation of subsets of atoms in a macromolecule to
assign structure to a binding site region or loop. For example,
SEA-HNCA-TROSY or SEA-HNCACB-TROSY spectra can be obtained for a
macromolecule in the presence and absence of a ligand to identify
atoms of the macromolecule that interact with the ligand and to
assign their structure. These experiments are particularly
attractive because .sup.13C.sup..alpha. and .sup.13C.sup..beta.
chemical shifts are characteristic of certain amino acid types, as
described in Cavanaugh et al., Protein NMR Spectroscopy: Principles
and Practice, Academic Press, San Diego Calif. (1996), and can
facilitate the assignment process. This can provide complete
backbone and .sup.13C.sup..beta. resonance assignments for most of
loop regions of the molecule and therefore of the binding site
region. In addition to providing structure determination for ligand
binding site regions, the methods can be used to determine dynamics
of a binding site. For example, .sup.15N spin relaxation
measurements can be used to obtain information on internal dynamics
of binding site loops as described in Cavanaugh et al., supra.
Combination of SEA-TROSY with .sup.15N spin relaxation measurements
can provide such determinations with larger macromolecules than
available with .sup.15N spin relaxation measurements using TROSY
only.
[0095] The methods can also be used to identify differences in the
interactions that mediate binding of different ligands at the same
binding site region or loop of a macromolecule. These differences
can be identified using methods similar to those described above
for comparing a macromolecule in the presence and absence of a
ligand except that the methods are carried out with a macromolecule
in the presence of a first and second ligand and the respective
spectra compared. Atoms that are differentially protected from
second molecule exchange by the two ligands can be identified as
having a stronger interaction with the first ligand than the second
ligand. For example, an atom that has a stronger signal due to
acquisition of a magnetized proton in the presence of a first
ligand when compared to a signal measured in the presence of a
second ligand, can be identified as being protected from a second
molecule to a greater extent by the second ligand. Thus the atom
can be identified as having a stronger interaction with the second
ligand than the first ligand. In cases where presence of the ligand
does not cause the binding site resonances to disappear entirely,
chemical shift perturbation can be detected. More specifically,
crosspeaks perturbed in chemical shift in the first spectrum
compared to the second spectrum can be used to identify binding
site residues or structural and functional characteristics of
binding site residues.
[0096] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE I
Detection of Solvent-Exposed amides Using SEA-TROSY
[0097] This example demonstrates the simplification of an NMR
spectrum of a large protein by selective observation of
solvent-exposed .sup.15N atoms on the protein. A SEA-TROSY
(Solvent-Exposed Amides with TROSY) spectrum was determined with a
uniformly .sup.15N/.sup.2H-labeled catalytic portion of the enzyme
cytochrome P450 reductase from rat liver (P450R). As a reference,
an .sup.15N, .sup.1H TROSY spectrum was also determined for
P450R.
[0098] P450R consists of residues 56-678 of the rat protein and has
a molecular weight of 71 kDa. The .sup.15N/.sup.2H labeled P450R
protein was expressed in E. coli grown in D.sub.2O on
.sup.15N-Amonium Chloride containing Celltone (Martek Biosciences
Corp., Columbia Md.) supplemented M9 minimal media in a bioflo
fermentor (New Brunswick Scientific, Edison N.J.). The
.sup.15N/.sup.2H labeled P450R protein was isolated from the
soluble fraction of the E. coli cells using anion exchange and
2',5'-ADP-affinity chromatography as described in Shen et al., J.
Biol. Chem. 264:7584-7589 (1989). The .sup.15N/.sup.2H labeled
P450R protein was stored in H.sub.2O such that the amides and
hydroxyls exchanged and became protonated leaving all other
non-exchanged positions deuterated. A sample containing 200 .mu.l
of a 0.5 mM concentration of the .sup.15N .sup.2H labeled P450R at
pH 7.5 was used for NMR spectroscopy measurements as described
below.
[0099] The SEA-TROSY spectrum was obtained with the pulse sequence
shown in FIG. 2a with a .tau..sub.m of 100 ms. The TROSY spectrum
was obtained with the pulse sequence shown in FIG. 2a without the
SEA element shown in the dashed box. For the pulse sequences shown
in FIG. 2, narrow and wide bars represent 90 and 180 degree
radiofrequency (rf) pulses, respectively. Unless specified
otherwise, pulse phases are along the x-axis. The pulsed field
gradients were 500 .mu.s in duration with strengths of g.sub.1=20
G/cm, g.sub.2=30 G/cm, g.sub.3=40 G/cm, g.sub.4=15 G/cm and
g.sub.5=55 G/cm. The delay .tau. was set to 2.7 ms. The phase cycle
was .phi..sub.1=y, -y, -x, x; .phi..sub.2=y; .phi..sub.3=x;
.psi..sub.rec=x, -x, -y, y. A phase sensitive spectrum in the
.sup.15N dimension was obtained by recording a second FID for each
t2 value, with .phi..sub.1=-y, y, -x, x, .phi..sub.2=-y and
.phi..sub.3=-x, and the data processed as described in Pervushin et
al., J. Biomol. NMR 12:345-348 (1998). Residual water suppression
was obtained with a WATERGATE sequence implemented with a 3-9-19
composite pulse as described in Sklenar et al., J. Magn. Reson.
102:241-244 (1993). The part of the sequence occurring before
.tau..sub.m in the pulse sequence shown in FIG. 2a is the .sup.15N
double filter.
[0100] The spectra were recorded at 303.degree. K. on a Bruker
DRX700 (Bruker AG, Fallanden, Switzerland) operating at 700 MHz
.sup.1H frequency, equipped with four rf channels, a triple
resonance probe with a shielded triple axis gradient coil and
deuterium switch box for deuterium decoupling. For each spectrum,
128 (t1)*1024(t2) complex points were recorded. Spectra were
acquired and processed with XWINNMR (Bruker AG). Spectra were
analyzed using XEASY as described in Bartels et al., J. Biomol. NMR
5:1-10 (1995).
[0101] As shown in the TROSY panel on the left side of FIG. 3, only
about 400 out of 630 expected crosspeaks could be resolved in the
spectrum. Most of the spectrum was characterized by severe
resonance overlap, precluding resonance assignments.
[0102] In contrast, the spectrum from the SEA-TROSY experiment at
100 ms mixing time, shown in the right panel of FIG. 3 is
dramatically simplified with significantly reduced resonance
overlap. The level of spectral complexity and amount of resonance
overlap observed in the spectrum was found to change with mixing
time .tau..sub.m, so that increased mixing time led to increased
complexity and resonance overlap.
EXAMPLE II
Combination of the SEA Element with Other NMR Pulse Sequences
[0103] This example demonstrates assignment of NMR signals to
specific regions in a large protein structure using spectra
obtained with a combination of the SEA pulse sequence and other NMR
pulse sequences. A 3D SEA-HNCA-TROSY spectrum was acquired for
.sup.15N/.sup.3C/.sup.2H labeled P450R and is shown in FIGS. 4a to
4c. A 3D HNCA-TROSY spectrum can be used to determine
connectivities between .sup.13C.sup..alpha. atoms in a protein
backbone as described in Salzmann et al., Proc. Natl. Acad. Sci.
USA 95:13585-13590 (1998).
[0104] The .sup.15N/.sup.13C/.sup.2H P450R was expressed and
isolated as described in Example I except that .sup.13C was added
to the growth medium in the form of (U-.sup.13C)glucose. A sample
containing 200 .mu.l of a 0.5 mM concentration of the
.sup.15N/.sup.13C/.sup.2H labeled P450R at pH 7.5 was used for NMR
spectroscopy measurements as described below.
[0105] The spectra were acquired, processed and analyzed as
described in Example I using the pulse sequence shown in FIG. 2b.
The SEA element was combined with a 3D HNCA-TROSY pulse scheme
(Saltzman et al., supra (1998)) to produce the 3D SEA-HNCA-TROSY
pulse scheme shown in FIG. 2b. .sup.13C.sup..alpha. 180 degree
pulses are band-selective REBURP pulses as described in Geen and
Freeman, J. Magn. Reson. 93:93-96 (1991). The REBURP pulses have
250 .mu.s duration centered at 53 ppm and are designed to
selectively excite the .sup.13C.sup..alpha. region without exciting
the .sup.13CO region (.about.177 ppm). This was done to avoid
losses of magnetization due to .sup.15--.sup.13CO .sup.1J coupling
constants (.about.15 Hz). .sup.13CO decoupling pulses were
off-resonance, Gaussian shaped pulses of 120 .mu.s duration shifted
to 177 ppm. The delays were .tau.=2.7 ms; .tau.=11 ms. The phase
cycle was .phi..sub.1=4 (x), 4 (-x); .phi..sub.2=y, -y, -x, x;
.phi..sub.3=y; .phi..sub.4=x; .psi..sub.rec=x, -x, -y, y, -x, x, y,
-y. A phase-sensitive spectrum in the .sup.15N dimension was
obtained by recording a second FID for each t2 value, with
.phi..sub.2=2(x), 2(-x); .phi..sub.3=x, -x, and the data processed
as described in Pervushin et al., supra. States-TPPI quadrature
detection in the .sup.13C.sup..alpha. dimension was achieved by
incrementing .phi..sub.1 as described in Marion et al., J. Magn.
Reson. 85:393-399 (1989). The pulsed field gradients were 500 .mu.s
in duration with strengths of g.sub.1=20 G/cm, g.sub.2=30 G/cm,
g.sub.3=40 G/cm, g.sub.4=15 G/cm, g.sub.5=40 G/cm, g.sub.6=15 G/cm,
g.sub.7=55 G/cm. .sup.2H decoupling during .sup.13C.sup..alpha.
evolution was achieved with a WALTZ-16 composite pulse, as
described in Shaka et al., J. Magn. Reson. 52:335-338 (1983), at a
field strength of 2.5 kHz. Suppression of residual water was
achieved with a WATERGATE sequence using a 3-9-19 composite pulse
as described in Sklenar et al., supra.
[0106] Combination of the SEA element with the 3D HNCA-TROSY
element provided a 3D SEA-HNCA-TROSY which was simplified compared
to the same spectrum acquired without the SEA element. As shown in
FIGS. 4a to 4c, the simplified spectrum could be used to assign
resonances in several loop regions of P450R. A stretch of a loop
consisting of residues Ala 235 to Glu 238 could be obtained via the
sequential .sup.13C(i)/.sup.13C(i-1) connectivities indicated by
the dashed line in FIG. 4a and the unique chemical shift pattern of
this stretch of residues versus the primary structure of P450R.
Using a similar approach loops consisting of Ala 500 to Asn 503 and
Asp 632 to Ala 633 were sequentially assigned as shown in FIGS. 4b
and 4c.
[0107] Because solvent-exposed residues are often located in loops,
these can be easily assigned via .sup.13C.sup..alpha.
connectivities, as in Cavanaugh et al., supra. Several
solvent-exposed loop regions in P450R were implicated in its
function. Assignments of chemical shifts for these loops using the
above described methods can be used to characterize their
involvement in protein catalysis, protein-protein interactions and
allosteric modulation, that determine the complex function of this
ubiquitous protein including functions described in Wang et al.,
Biochemistry 94:8411-8416 (1997). For example, the small loop of
residues Gly 631 to Ala 633 is of particular interest because it is
adjacent to the catalytic residue Cys 630 and presumably makes
contact with the third cofactor NADP as described in Wang et al.,
supra, and Shen et al., J. Biol. Chem. 274:5391-5398 (1999).
[0108] The publications cited herein are hereby incorporated by
reference. The term "comprising" is intended herein to be
open-ended, including not only the recited elements, but further
encompassing any additional elements.
[0109] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *