U.S. patent application number 10/293580 was filed with the patent office on 2003-09-11 for fluorescent protein sensors of post-translational modifications.
This patent application is currently assigned to Aurora Biosciences Corporation. Invention is credited to Cubitt, Andrew B..
Application Number | 20030170767 10/293580 |
Document ID | / |
Family ID | 22438839 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170767 |
Kind Code |
A1 |
Cubitt, Andrew B. |
September 11, 2003 |
Fluorescent protein sensors of post-translational modifications
Abstract
The present invention includes a fluorescent compound that can
detect an activity. such as an enzymatic activity, and exhibits
quenching. The fluorescent compound can include a fluorescent
protein, such as an Aequorea-related green fluorescent protein. The
fluorescent compound can include a substrate site for an enzymatic
activity such as a kinase activity, a phosphatase activity, a
protease activity, and a glycosylase activity The fluorescent
compound of the present invention can be used to detect such
enzymatic activities in samples, such as biological samples,
including cells. The present invention also includes nucleic acids
that encode the fluorescent compounds of the present inventions,
and cells that include such nucleic acids or fluorescent
compounds.
Inventors: |
Cubitt, Andrew B.; (San
Diego, CA) |
Correspondence
Address: |
GARY CARY WARE & FRIENDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
Aurora Biosciences
Corporation
|
Family ID: |
22438839 |
Appl. No.: |
10/293580 |
Filed: |
November 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10293580 |
Nov 12, 2002 |
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09129192 |
Jul 24, 1998 |
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6495664 |
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Current U.S.
Class: |
435/15 ; 435/23;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/43595
20130101 |
Class at
Publication: |
435/15 ;
435/69.1; 435/320.1; 435/325; 435/23; 530/350; 536/23.5 |
International
Class: |
C12Q 001/48; C12Q
001/37; C07H 021/04; C12P 021/02; C12N 005/06; C07K 014/435 |
Claims
I claim:
1. A fluorescent compound for detecting an activity, comprising: a
fluorescent protein moiety, and at least one exogenous substrate
recognition motif for an activity, wherein said fluorescent protein
moiety can be converted from a first state to a second state in
response to said activity, further wherein said fluorescent
compound exhibits at least one different fluorescent property in
said first state and said second state under quenching
conditions.
2. The fluorescent compound of claim 1, wherein said activity is an
enzymatic activity.
3. The fluorescent compound of claim 2, wherein said enzymatic
activity is selected from the group consisting of a kinase
activity, a phosphatase activity, a protease activity, a
glycosylation activity, and a farnesyl transferase activity.
4. The fluorescent compound of claim 1, wherein said fluorescent
protein moiety comprises an Aequorea-related fluorescent
protein.
5. The fluorescent compound of claim 1, wherein said fluorescent
protein moiety comprises a phosphorylation recognition motif for a
protein kinase is a serine/threonine specific protein kinase.
6. The fluorescent compound of claim 1, wherein said fluorescent
protein moiety comprises a phosphorylation recognition motif for a
protein kinase selected from the group consisting of protein kinase
A, a cGMP-dependent protein kinase, protein kinase C,
Ca.sup.2+/calmodulin-dependent protein kinase I,
Ca.sup.2+/calmodulin-dependent protein kinase II, and MAP kinase
activated protein kinase.
7. The fluorescent compound of claim 4, wherein said
Aequorea-related fluorescent protein moiety comprises the mutations
in GFP mutant K8.
8. The fluorescent compound of claim 4, wherein said at least one
exogenous substrate recognition motif for an activity is within the
first 20 amino acids of the amino terminus of said Aequorea-related
fluorescent protein moiety.
9. The fluorescent compound of claim 4, wherein said at least one
exogenous substrate recognition motif for an enzymatic activity is
within the first 10 amino acids of the amino terminus of said
Aequorea-related fluorescent protein moiety.
10. The fluorescent compound of claim 1, wherein said quenching
conditions is acid quenching.
11. The fluorescent compound of claim 4, wherein said
Aequorea-related fluorescent protein moiety is membrane
associated.
12. The fluorescent compound of claim 11, wherein said
Aequorea-related fluorescent moiety comprises a poly-Lys
region.
13. The fluorescent compound of claim 4, wherein said
Aequorea-related fluorescent protein moiety comprises a
protein-protein interaction domain.
14. The fluorescent compound of claim 4, wherein said
Aequorea-related fluorescent moiety is membrane bound.
15. A nucleic acid molecule coding for the expression of a
fluorescent compound, wherein said fluorescent compound comprises a
fluorescent protein moiety, and at least one exogenous substrate
motif for an activity, further wherein said fluorescent protein
moiety can be converted from a first state to a second state by an
activity, further wherein said first state and said second state
can be differentiated under quenching conditions.
16. A cell, comprising: a nucleic acid molecule coding for the
expression of a fluorescent compound, wherein said fluorescent
compound comprises a fluorescent protein moiety, and at least one
exogenous substrate motif for an activity, wherein said fluorescent
protein moiety can be converted from a first state to a second
state by an activity, further wherein said first state and said
second state can be differentiated under quenching conditions.
17. A method for determining whether a sample contains an activity,
comprising: contacting a sample with a fluorescent compound,
wherein said fluorescent compound comprises a fluorescent protein
moiety, and at least one exogenous substrate motif for an activity,
further wherein said fluorescent protein moiety can be converted
from a first state to a second state by an activity, exciting said
fluorescent compound, and measuring the amount of emission from
said fluorescent compound.
18. The method of claim 17, further comprising the step of
comparing the amount of emission measured from said fluorescent
compound with the emission from a control sample.
19. The method of claim 18, wherein said enzymatic activity is
selected from the group consisting of a kinase activity, a
phosphatase activity, a protease activity, a glycosylase activity,
and a farnsyl transferase activity.
20. The method of claim 19, wherein said quenching is acid
quenching.
21. A method for determining whether a cell exhibits an activity
comprising the steps of: exciting a transfected host cell
comprising a recombinant nucleic acid molecule, wherein said
recombinant nucleic acid molecule comprises at least one expression
control sequence operatively linked to a nucleic acid sequence
coding for the expression of a fluorescent compound, wherein said
fluorescent compound comprises a fluorescent protein moiety, and a
substrate motif for an activity, further wherein said fluorescent
compound can be converted from a first state to a second state in
the presence of said activity, further wherein said first state and
said second state and be differentiated under quenching conditions,
and measuring the emission from said fluorescent compound.
22. The method of claim 21, wherein said enzymatic activity is
selected from the group consisting of a kinase activity, a
phosphatase activity, a protease activity, a glycosylase activity,
and a farnsyl transferase activity.
23. A method for determining whether a sample contains an enzymatic
activity, comprising: a) contacting a sample with a fluorescent
compound, wherein said fluorescent compound comprises an
Aequorea-related fluorescent protein moiety and an exogenous
substrate motif for an enzymatic activity, further wherein said
fluorescent compound can be converted from a first state to a
second state by said enzymatic activity, further wherein said first
state and said second state can be differentiated under quenching
conditions, b) exciting said fluorescent compound, and c) measuring
the amount of fluorescence emitted from said sample, whereby the
amount of quenching that is consistent with the presence of said
enzyme activity indicates the presence of said enzyme activity in
said sample.
24. A compound identified by the method comprising the steps of: a)
contacting a sample with a fluorescent compound, wherein said
fluorescent compound comprises a fluorescent protein moiety and an
exogenous substrate motif for an activity, further wherein said
fluorescent compound can be converted from a first state to a
second state by said activity, further wherein said first state and
said second state can be differentiated under quenching conditions,
b) exciting said fluorescent compound, and c) measuring the amount
of fluorescence emitted from said sample.
Description
TECHNICAL FIELD
[0001] The present invention generally relates to compositions and
methods for the detection of activities, such as enzymatic
activities, using fluorescent compounds that are modified by the
activity such that they exhibit a change in their sensitivity to
quenching.
BACKGROUND
[0002] Fluorescent compounds have been used in the art to detect a
wide variety of biological phenomenon, such as changes in ion
concentration, specific binding reactions. subcellular
localization, and enzymatic reactions. In the case of detecting
changes in ion concentration, specific binding reactions, and
subcellular localization, the fluorescent compound is used as a
label to detect such specific binding or localization. In some
cases, the fluorescence of the fluorescent compound is altered
after an enzyme has acted on the fluorescent compound or a molecule
binds with the fluorescent compound. For example, the activity of
beta-galactosidase on the substrate fluorescein
di-beta-D-galactopyranoside causes an increase in fluorescence of
the substrate (Molecular Probes Catalogue, Sixth Edition, p. 208
(1996)). Likewise, the action of beta-lactamase on CCF2-AM causes
the compound to change fluorescence from green to blue due to an
uncoupling of fluorescence resonance energy transfer (FPET) (Tsien
and Zlokamik, WO 96/30540, published Oct. 3, 1996). Protease
activity can also be detected by the action of a protease on a
fluorescent compound that uncouples FRET of the fluorescent
compound (Tsien et al., WO 97/28261, published Aug. 7, 1997).
[0003] The detection of enzymatic reactions is important for the
study of biological phenomenon, cellular biology, medical
diagnostics, and drug discovery. Several classes of enzymes have
been implicated in disease states, such as proteases for HIV and
kinases for cancer. Drug discovery preferably uses living cells to
detect compounds that can alter the activity enzymes involved in
such disease states. However, ex vivo methods can also be used in
drug discovery
[0004] Protein kinases and phosphatases have particularly been
recognized as one of the more important general mechanism of
cellular regulation. Protein phosphorylation commonly occurs on
three major amino acids, tyrosine, serine or threonine. Changes in
the phosphorylation state of these amino acids within proteins can
regulate many aspects of cellular metabolism, regulation, grown and
differentiation. Changes in the phosphorylation state of proteins,
mediated through phosphorylation by kinases, or dephosphoryation by
phosphatases, is a common mechanism through which cell surface
signaling pathways transmit and integrate information into the
nucleus. Given their key role in cellular regulation, it is not
surprising that defects in protein kinases and phosphatases have
been implicated in many disease states and conditions. For example,
the over-expression of cellular tyrosine kinases such as the EGF or
PDGF receptors, or the mutation of tyrosine kinases to produce
constitutively active forms (oncogenes) occurs in many cancer cells
(Durker et al. Nature Medicine 2:561-556 (1996)). Protein tyrosine
kinases are also implicated in inflammatory signals, and defective
Thr/Ser kinase genes have been demonstrated to be implicated in
several diseases such as myotonic dystrophy, cancer and Alzheimer's
disease (Sanpei et al., Biochem. Biophys. Res. Commun. 212:341-346
(1995); Sperger et al., Neurosci Lett. 197:149-153 (1995); Grammas
et al., Neurobiology of Aging, 16:563-569 (1995); Govani et al.,
Ann. N.Y. Acad. Sci. 777:332-337 (1996)).
[0005] The involvement of proteases, protein kinases, protein
phosphatases, and other classes of enzymes in disease states makes
them attractive targets for the therapeutic intervention of drugs.
In fact, many clinically useful drugs act on protein kinases or
phosphatases. Examples include cyclosporin A, a potent
immunosuppresent that binds to cyclophilin. This complex binds to
the Ca.sup.++/calmodulin-dependent protein phosphatase type 2B
(calcineurin), inhibiting its activity, and hence the activation of
T cells. Inhibitors of protein kinase C are in clinical trials as
therapeutic agents for the treatment of cancer (Clin. Cancer Res.
1:113-122 (1995)) as are inhibitors of cyclin dependent kinase (J.
Mol. Med. 73:509-514 (1995)).
[0006] The number of known enzymes, such as kinases and
phosphatases, are growing rapidly as the influence of genomic
programs to identify the molecular basis for diseases have
increased in size and scope. These studies are likely to implicate
many more genes that encode enzymes that are involved in the
development and propagation of diseases in the future, thereby
making them attractive targets for drug discovery. However, current
methods of measuring enzyme activity, such as protein
phosphorylation and dephosphorylation, have many disadvantages
which prevents or limits the ability to rapidly screen for drugs
using miniaturized automated formats of many thousands of
compounds. In the case of phosphatases and kinases, this is because
current methods rely on the incorporation and measurement of
.sup.32P into the protein substrates of interest. In whole cells
this necessitates the use of high levels of radioactivity to
efficiently label the cellular ATP pool and to ensure that the
target protein is efficiently labeled with radioactivity. After
incubation with test drugs, the cells must be lysed and the protein
of interest purified to determine its relative degree of
phosphorylation. This method requires high numbers of cell, long
preincubation times, careful manipulation, and washing steps to
avoid artifactal phosphorylation or dephosphorylation. Furthermore,
this approach requires purification of the target protein, and
final radioactive incorporation into target proteins is usually
very low, giving the assay poor sensitivity. Alternative assay
methods, such as those based on phosphorylation-specific antibodies
using ELISA-type approaches, involve the difficulty of producing
antibodies that distinguish between phosphorylated and
non-phosphorylated proteins, and the requirement for cell lysis,
multiple incubations, and washing stages which are time consuming,
complex to automate, and potentially susceptible to artifacts.
[0007] Fluorescent molecules are attractive as reporter molecules
in many assay systems because of their high sensitivity and ease of
quantification. Recently, fluorescent proteins have been the focus
of much attention because they can be produced in vivo by
biological systems and can be used to trace and monitor
intracellular event without the need to be introduced into the cell
through microinjection or permeabilization. The green fluorescent
protein of Aequorea Victoria is particularly interesting as a
fluorescent indicator protein. A cDNA for the protein has been
cloned (Prasher et al., Gene 111:229-233 (1992)). Not only can the
primary amino acid sequence of the protein be expressed from the
cDNA, but the expressed protein can fluoresce in cells in vivo.
[0008] Fluorescent proteins have been used as markers of gene
expression tracers of cell lineage, and as fusion tags to monitor
protein localization within living cells (Rizzuto et al., Current
Biol. 6:183-188 (1996)); Cubitt et al., TIBS 20:448-455 (1995);
U.S. Pat. No. 5,625,048 to Tsien et al, issued Apr. 29, 1997).
Furthermore, mutant versions of green fluorescent protein have been
identified that exhibit altered fluorescence characteristics,
including altered excitation and emission maxima, as well as
excitation and emission spectra of different shapes. (Heim, Proc.
Natl. Acad. Sci. USA 91:12501-12504 (1994); Heim et al., Nature
373:663-665 (1995); U.S. Pat. No. 5,625,048, Tsien et al., issued
Apr. 29, 1997; WO 97/28261 to Tsien et al, published Aug. 7, 1997;
PCT/US 97/12400 to Tsien, filed Jul. 16, 1997; and PCT/US 97/14593
by Tsien, filed Aug. 15, 1997). These proteins add variety and
utility to the arsenal of biologically based fluorescent
indicators.
[0009] There is thus a need for assays for enzymes, such as those
involved in protein phosphorylation, that are sensitive, simple to
use, useful in living cells, and adaptable to high throughput
screening methods. Preferably, such assays would not utilize
radioactive materials so that the assays would be safe and not
generate hazardous wastes. The present invention addresses these
needs, and provides additional benefits as well.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 depicts the nucleotide sequence (SEQ ID NO:1) and
deduced amino acid sequence (SEQ ID NO:2) of wild-type Aequorea
green fluorescent protein.
[0011] FIG. 2 depicts a list of the positions and amino acid
changes made for phosphorylation mutants made more than fifteen
amino acids in the primary sequence from the N-terminus, as
compared to FIG. 1. Amino acids underlined represent the
phosphorylation motif, amino acids in brackets represent wild type
sequence at those positions.
[0012] FIG. 3 depicts the bacterial expression plasmid pRSET
(Invitrogen) containing a region encoding GFP that is fused in
frame with nucleotides encoding an N-terminal polyhistidine
tag.
[0013] FIG. 4 depicts a duel expression vector having expression
control sequences operably linked to sequences encoding for the
expression of protein kinase A catalytic subunit (PKA cat) upstream
from sequences coding for the expression of a fluorescent protein
substrate.
[0014] FIG. 5 depicts the effect of incubation time on the
stability of GFP mutant K8 fluorescence after termination of a
kinase reaction with 100 mM acetate buffer, 100 mM NaCl, 25 mM
beta-glycerol phosphate pH 5.0.
[0015] FIG. 6 depicts the effects of quenching on the fluorescent
properties of the GFP mutant either after phosphorylation by
protein kinase A or in the absence of protein kinase A.
[0016] FIG. 7 depicts the kinetics of phosphorylation of a GFP
having a kinase motif
[0017] FIG. 8 depicts the determination of the dose dependent
inhibition of PKA by known inhibitors of that enzyme using GFB
mutant KS.
[0018] FIG. 9 depicts the phosphorylation of GFP with and without a
membrane association motif
SUMMARY
[0019] The present invention recognizes that fluorescent compounds,
such as fluorescent proteins, can exist in at least two states and
that the fluorescent properties of these two states can be
different, preferably after exposure of the fluorescent compound to
quenching conditions. Particularly, the stability of the
fluorescence in one state can be different from the stability of
the fluorescence in the second state, which can be detected by
their susceptibility to quenching. The first and second states of
the fluorescent compound can be caused by the action of a chemical
or enzyme. Thus, the present invention recognizes that such
fluorescent compounds can be used to detect various chemical or
enzymatic activities in a sample. The present invention recognizes
that fluorescent compounds can be fluorescent proteins that can be
expressed in cells. Thus, the fluorescent proteins can be used as
in vivo monitors of enzymatic activity (intracellular or
extracellular) and can be used to screen compounds for drugs that
modulate (i.e., increase or decrease the activity) an enzymatic
activity. The present invention also provides nucleic acid
molecules that encode fluorescent proteins that exhibit
quenching.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Definitions
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this. invention belongs.
Generally, the nomenclature used herein, and the laboratory
procedures in cell culture, molecular genetics, and nucleic acid
chemistry and hybridization described below, are those well known
and commonly employed in the art. Standard techniques are used for
recombinant nucleic acid methods, polynucleotide synthesis, and
microbial culture and transformation (e.g., electroporation, and
lipofection). Generally, enzymatic reactions and purification steps
are performed according to the manufacturer's specifications when
kits or purchased reagents or materials are used. The techniques
and procedures are generally performed according to conventional
methods in the art and various general references (see generally,
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed.
(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.) which are provided throughout this document. The nomenclature
and laboratory procedures used herein are those well known and
commonly employed in the art. As used throughout the disclosure,
the following terms, unless otherwise indicated, shall be
understood to have the following meanings.
[0022] "Quenching" or "quenching conditions," as used herein, means
conditions that can cause a change in at least one fluorescent
property of a fluorescent compound in a first state as compared to
a second state. Quenching can be used, for example, to detect or
measure the presence or concentration of the fluorescent compound
in the first state or second state. Quenching conditions can
include at least one quenching agent.
[0023] A first state or a second state of a fluorescent compound
means that the fluorescent compound exists in at lest two states,
wherein the different states have different fluorescent properties
that can be detected by quenching. The first state can differ from
the second state, for example, by the covalent attachment of
moieties to the fluorescent compound, the binding or association of
moieties to the fluorescent compound, the cleavage or disruption of
covalent or non-covalent bonds or interactions on or within the
fluorescent compound, the binding or association of the fluorescent
compound to other moieties or itself, or a change in the
conformation of the fluorescent compound as a result of the
presence of an activity.
[0024] "Quenching agent," as used herein, can be any chemical,
compound or biological molecule that can cause quenching of a
fluorescent compound, either alone or in combination with other
agents or factors
[0025] A "fluorescent compound," as used herein, can be any
fluorescent chemical or compound that can exhibit at least one
different fluorescent property in a first state and a second state
under quenching, conditions. For example, fluorescent compounds can
be small aromatic compounds such as fluorescein or rhodamine, or a
weakly or non-fluorescent compound such as, for example
carbohydrates, lipids, proteins, peptides, polypeptides, nucleic
acids that has been labeled with a highly fluorescent compound or
combinations thereof. For example, a fluorescent compound can be a
fluorescent protein moiety. A fluorescent compound, such as those
comprising a fluorescent protein moiety, can be soluble, membrane
bound, or membrane associated. Membrane bound versions of soluble
fluorescent protein moieties can be made by adding, for example,
hydrophobic regions such as signal sequences or hydrophobic
moieties as they are known in the art using established methods.
Likewise, membrane associated versions of soluble fluorescent
protein moieties can be made by adding, for example, membrane
association motifs, such as poly-Lys, to such fluorescent protein
moieties using established methods.
[0026] A "fluorescent protein moiety" means any protein or fragment
thereof capable of fluorescence when excited with appropriate
electromagnetic radiation. This includes fluorescent proteins whose
amino acid sequences are either naturally occurring or engineered
(i.e., analogs) and proteins that have been modified to be
fluorescent, such as by the addition of a fluorescence compound,
such as fluorescein, rhodamine, Cy3-5, Cy-PE, lucifer yellow,
C6-NBD, Dio-Cn(3), FITC, Biodipy-FL, eosin, propidium iodide,
tetramethyl rhodamine B, Dil-Cn-(3), Lissamine Rhodamine B, Texas
Red, Allophycocyanin, Dil-Cy-5, and squaranes by methods known in
the art. For fluorescent compounds, see Molecular Probes Catalogue
(1998), U.S. Pat. No. 5,631,169, issued May 20, 1997, U.S. Pat. No.
5,145,774, issued Sep. 8, 1992, and world wide web site
http://optics.jct.ac.il/.about.aryeh/Confocal/fluoreochromes (Jul.
6, 1998) Many cnidarians use green fluorescent proteins ("GFPs") as
energy-transfer acceptors in bioluminescence. A "green fluorescent
protein," as used herein, is a protein that fluoresces green light.
Similarly, "blue fluorescent proteins" fluoresce blue light and
"red fluorescent proteins" fluoresce red light. GFPs have been
isolated from the Pacific Northwest jellyfish, Aequorea Victoria,
the sea pansy, Renilla reniformis, and Phialidium gregarium (W. W.
Ward et al., Photochem. Photoobiol, 35:803-808 (1982); Levine et
al, Comp. Biochem. Physiol. 72B:77-85 (1982); and Roth,
Purification and Protease Susceptibility of the Green-Fluorescent
Protein of Aequorea Aequorea With a Note on Halistaura,
Dissertation, Rutgers, The State University of New Jersey, New
Brunswick, N.J. (1985)). GFPs have also been engineered to be blue
fluorescent proteins and yellow fluorescent proteins (U.S. Pat. No.
5,625,048 to Tsien et al., issued Apr. 29, 1997; WO 97/28261 to
Tsien et al., filed Jul. 16, 1997; PCT/US 97/14593 to Tsien et al.,
filed Aug. 15, 1997; WO 97/28261 to Tsien, published Aug. 7, 1997;
and WO 96/23810 to Tsien et al., published Aug. 18, 1996).
[0027] An "Aequorea-related fluorescent protein" means any protein
that has any contiguous sequence of 150 amino acids that has at
least 85% sequence identity with an amino acid sequence, either
contiguous or non-contiguous, from the 238 amino acid wild-type
Aequorea green fluorescent protein of SEQ ID NO:2. More preferably,
a fluorescent protein is an Aequorea-related fluorescent protein if
any contiguous sequence of 200 amino acids of the protein has at
least 95% sequence identity with an amino acid sequence, either
contiguous or non-contiguous, from the wild type Aequorea green
fluorescent protein of SEQ ID NO:2. Similarly, the protein can be
related to Renilla or Phialidium wild-type fluorescent proteins
using the same standards.
[0028] A variety of Aequorea-related fluorescent proteins have been
engineered by modifying the amino acid sequence of a naturally
occurring GFP from Aequorea victoria (D. C. Prasher et al, Gene,
111:229-233 (1992); Heim et al. Proc. Natl. Acad. Sci. USA
91:12501-12504 (1994); U.S. Pat. No. 5,625,048, issued Apr. 29,
1997 to Tsien et al.; WO 96/23810 to Tsien, published Aug. 8, 1996;
and PCT application PCT/US97/14593 to Tsien et al, filed Aug. 15,
1997) and have useful excitation and emission spectra.
[0029] A "substrate site for an activity" means a locus that is a
substrate for an activity, such as an enzymatic activity. The locus
can be any structure, such as an amino acid, chemical group or
ionic or covalent bond. Such substrate site for an activity can be
part of a substrate recognition motif that is recognized by an
activity. For example, the site of phosphorylation within a protein
is the actual amino acid modified by a phosphatase or kinase, and
the site of phosphorylation can be within a phosphorylation
recognition motif. A fluorescent compound or fluorescent protein
moiety can have at least one substrate recognition motif, which can
have at least one substrate site for an activity. Substrate
recognition motifs that are recognized by enzymatic activities are
known in the art, such as for proteases, kinases, phosphatases,
glycosylases, or transferases (such as famsyl transferases), or any
other type of enzyme.
[0030] A "substrate recognition motif for an activity" can be any
structure or sequence that is recognized by an enzyme that directs
or helps in the enzymatic modification of the substrate by the
enzyme. The substrate recognition motif for an activity can be
within. close to, or part of the structure, such as amino acid
residue or residues, that are modified by the activity, such as an
enzyme activity, (such as the substrate site for an activity). For
example, the sequence surrounding a protein kinase A
phosphorylation site plays a significant role in controlling how
efficiently the site is modified. Also, protein-protein interaction
domains and protein localization domains can control the efficiency
of enzymatic modifications of a substrate, such as a protein
substrate, and are particularly important within cells (see, Pawson
et al., Science 278:2075-2080 (1997). These protein-protein
interaction domains and protein localization domains can be distal
from the substrate recognition motif and play a role in substrate
recognition.
[0031] A "fluorescent protein substrate" is a substrate for an
activity, such as an enzymatic activity, that comprises a
fluorescent compound that comprises a fluorescent protein moiety
and at least one substrate site for an activity.
[0032] As used herein the term "phosphorylation recognition motif
for a protein kinase" refers to an amino acid sequence that is
recognized by a protein kinase for the attachment of a phosphate
moiety. The phosphorylation recognition motif for a protein kinase
can be a site recognized by, for example, protein kinase A, a
cGMP-dependent protein kinase, protein kinase C,
Ca.sup.2+/calmodulin-depending protein kinase I,
Ca.sup.2+/calmodulin-dependent protein kinase II or MAP kinase
activated protein kinase type I, and isoforms or allelic variants
thereof
[0033] As used herein, "fluorescent property" refers to the molar
extinction coefficient at an appropriate excitation wavelength, the
fluorescent quantum efficiency, the shape of the excitation
spectrum or emission spectrum, the excitation wavelength maximum or
emission wavelength maximum, the ratio of excitation amplitudes at
two different wavelengths, the ratio of emission amplitudes at two
different wavelengths, the excited state lifetime, the fluorescent
anisotropy or any other measurable property of a fluorescent
compound. A measurable difference in any one of these properties in
response to a quenching agent or under quenching conditions between
a first state and a second state of a fluorescent compound suffices
for the utility of the fluorescent compounds of the invention.
[0034] A difference in a fluorescent property of a fluorescent
compound can be measured by determining the amount of any
quantitative fluorescent property, e.g., the amount of fluorescence
at a particular wavelength, or the integral of fluorescence of the
emission spectrum. Determining ratios of excitation amplitude or
emission amplitude at two different wavelengths ("excitation
amplitude ratioing" and "emission amplitude ratioing,"
respectively) are particularly advantageous because the ratioing
process provides an internal reference an cancels out variations in
the absolute brightness of the excitation source, the sensitivity
of the detector, and light scattering or quenching by the sample.
Furthermore, if a change in a fluorescent compound from a first
state to a second state changes the fluorescent compound's ratio of
excitation or emission amplitudes at two different wavelengths,
then such ratios measure the extent of the first state and second
state independent of the absolute quantity of the fluorescent
compound.
INTRODUCTION
[0035] The present invention recognizes that fluorescent compounds,
such as fluorescent proteins, can exist in at least two states and
that the fluorescent properties of these two states can be
different, preferably under quenching conditions, and reflect
different biochemical or chemical characteristics of the
fluorescent compounds. Particularly, the stability of the
fluorescence in one state can be different from the stability of
the fluorescence in the second state, which can be detected by a
sensitivity to quenching The conversion of a first state to a
second state of the fluorescent compound can be caused by the
action of a chemical or enzyme, such as a protease, kinase,
phosphatase, glycosylase, transferase such as protein prenyl
transferase, or any other enzyme that can modify the fluorescent
compound. Binding of moieties or hybridization (in the case of
nucleic acids such as DNA or RNA) can also alter the fluorescence
properties of a fluorescent compound after quenching. Thus, the
present invention recognizes that such fluorescent compounds can be
used to detect various enzymatic activities in a sample, such as in
a cell, a cell culture, a cell extract, conditioned medium, or in
an array. Such hybridization or binding can occur and be detected
in high-density arrays or on gene chips such as they are known in
the art (See, Johnson. Curr. Biol. 8:R171-4 (1998); Livache et al.,
Anal. Biochem. 255:188-194 (1998)). The present invention
recognizes that fluorescent compounds can be fluorescent proteins
and that these fluorescent proteins can be expressed in cells.
Thus, the fluorescent proteins can be used as in vivo monitors of
intracellular enzymatic activity and used to screen compounds for
drugs that modulate an enzymatic activity. The present invention
also provides nucleic acid molecules that encode fluorescent
proteins that exhibit quenching.
[0036] As a non-limiting introduction to the breath of the
invention, the invention includes several general and useful
aspects, including:
[0037] 1) A fluorescent compound for detecting an activity,
comprising a fluorescent protein and a substrate recognition motif
for an activity. wherein said fluorescent compound exhibits
quenching,
[0038] 2) A nucleic acid molecule coding for the expression of the
fluorescent compound in 1),
[0039] 3) A cell comprising the nucleic acid molecule of 2) or
fluorescent compound of I),
[0040] 4) A method for determining whether a sample contains an
activity, comprising contacting a sample with a fluorescent
compound of 1), exciting said fluorescent compound, and measuring
the amount of emission from said fluorescent compound under
quenching conditions, and
[0041] 5) A method for determining whether a cell exhibits an
activity comprising exciting a transfected host cell comprising a
recombinant nucleic acid molecule of 2) or fluorescent compound of
1). and measuring the emission from the fluorescent compound under
quenching conditions.
[0042] Fluorescent Compounds for Detecting an Activity.
[0043] The present invention provides fluorescent compounds for
detecting an activity, comprising: a fluorescent protein and at
least one substrate site for an activity, wherein said fluorescent
compound can exist in at lease two states that exhibit
quenching.
[0044] The sensitivity of the fluorescent compound to quenching is
influenced by a modification of the fluorescent compound from a
first state to a second state by an activity, such as an enzymatic
activity. Such modification of the fluorescent compound can
stabilize or destabilize the fluorescent compound by, for example,
changing the conformation of the fluorescent compound, the addition
of a moiety to the fluorescent compound, or the removal of a moiety
from the fluorescent compound. Exposure of the fluorescent compound
to chemicals or enzymes can cause such modifications.
[0045] For example, a ligand binding to a fluorescent compound can
alter a fluorescent property of the fluorescent compound, making
the fluorescent compound more or less sensitive to quenching.
Likewise, the association of moieties with a fluorescent compound
(by, for example, chemical or enzymatic reaction) or the
aggregation of fluorescent compounds., or hybridization of nucleic
acids, can alter at least one fluorescent property of a fluorescent
compound that can be detected by quenching. Furthermore, moieties
can be added to a fluorescent compound by covalent modification
that can result in an altered fluorescent property of the
fluorescent compound that can be detected by quenching. Likewise,
covalent bonds can be cleaved within the structure of the
fluorescent compound that can result in an altered fluorescent
property of the fluorescent compound that can be detected after
quenching.
[0046] Covalent bonds can be made or broken by well-known chemical
or enzymatic reactions. For example, peptide bonds can be
hydrolyzed by acidic conditions or by proteases. Also, phosphate
groups can be added to a fluorescent compound by kinases, and
removed by phosphatases. Other enzymatic reactions, such as
lipases, the addition or loss of lipid moieties such as farnesyl,
geranylgeranyl or phosphatidyl inositol groups, and the like can be
used to modify fluorescent compounds, which can in turn alter a
fluorescent property of the fluorescent compound that can be
measured by quenching.
[0047] A change in the number or distribution of disulfide bonds by
an alteration in the redox state thereof can also alter a
fluorescent property of a fluorescent compound. For example,
reducing agents, such as beta-mercaptoethanol, can alter the
oxidation state of disulfide bonds within a fluorescent compound
having such structures. The change in the structure of the
fluorescent compound caused by such treatment can cause a change in
a fluorescent property of the fluorescent compound that can be
detected after quenching.
[0048] When an activity can modify a fluorescent compound, the
fluorescent compound can comprise a naturally occurring substrate
recognition motif (for example, endogenous to the fluorescent
compound) for such enzymatic reactions, or such substrate
recognition motifs can be added or engineered into the fluorescent
compound (for example exogenous to the fluorescent compound). For
example, a fluorescent compound that is a protein can be engineered
to comprise a substrate recognition motif for a protease, protein
phosphatase, protein kinase, protein prenyltransferase,
glycosylase, or any other enzyme using methods known in the art.
For example, genetic engineering, chemical modification techniques,
or enzyme reactions can be used to add such substrate recognition
motifs to the amino- or carboxy-terminus of a fluorescent compound.
Alternatively, these techniques can be used to insert such
substrate recognition motifs within the structure of the
fluorescent compound.
[0049] A change in the tertiary structure of a fluorescent compound
by the addition or removal of a moiety by chemical or enzymatic
activity can also cause a change in a fluorescent property of a
fluorescent compound after quenching. For example, phosphorylation
of a fluorescent compound can lead to a change in its tertiary
structure through the creation of new or stronger interactions
between amino acid residues, which can result in stabilized
fluorescence that can result in increased resistance to quenching.
Such a change in sensitivity to quenching can subsequently be used
to measure the amount of fluorescent compound that has been
phosporylated, and hence the activity of the kinase can be detected
and/or measured. Such moieties can also destabilize the tertiary
structure of a fluorescent protein, which can result in
destabilized fluorescence under quenching conditions. Furthermore,
enzymatic activities such as proteases can alter the tertiary
structure of a fluorescent protein, which can also result in
destabilized fluorescence under quenching conditions. Furthermore,
the presence of an electrochemical, chemical or electrical gradient
or potential can also change a fluorescent property of a
fluorescent compound that can be detected through quenching.
[0050] Quenching agents can be used to detect the stabilization,
destabilization, or protection of a fluorescent property of a
fluorescent compound arising from an activity. For example,
fluorescent compounds such as fluorescent proteins can be
stabilized or destabilized by acidic conditions, basic conditions,
surfactants, organic solvents, chaotropic salts or agents,
anti-chaotropic salts or agents, reducing conditions or agents,
oxidizing conditions or agents, paramagnetic ions, enzymes,
collisional quenchers? temperature or any combination thereof.
[0051] Fluorescent compounds, such as fluorescent proteins, can be
made using methods known in the art. For example, fluorescent
proteins can be made by expressing nucleic acids that encode
fluorescent proteins, such as wild-type or mutant Aequorea green
fluorescent protein, or other fluorescent proteins such as those
from Renilla, in an appropriate cellular host (WO 96/2381 to Tsien
et al., published Aug. 18, 1996). Alternatively, proteins that are
otherwise not fluorescent can be made fluorescent by covalently
linking or binding a fluorophore, such as fluorescein, to the
protein using methods known in the art (U.S. Pat. No. 5,605,809 to
Kumoriya et al., issued Feb. 25, 1997).
[0052] Fluorescent compounds, such as fluorescent proteins, can
have structures that allow them to be altered from a first state to
a second state by, for example, binding of a ligand, post
translational modifications such as phosphorylation,
dephosphorylation, proteolysis, or glycosylation at specific sites.
Compounds can be modified to include such specific sites using
methods known in the art. For example, fluorescent proteins, such
as Aequorea green fluorescent protein, can be engineered to have
non-naturally occurring substrate recognition motifs, such as known
phosphorylation or protease motifs and sites. The phosphorylation
or proteolytic status of the fluorescent compound can alter the
stability of the compound such that the phosphorylated,
unphosphorylated, or proteolytic states of the fluorescent compound
can be detected or measured using quenching.
[0053] Optimal alignment of sequences for aligning a comparison
window may be conducted by the local homology algorithm of Smith
and Waterman (Adv. Appl. Math, 2:482 (1981)) by the homology
alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48-:443
(1970)) by the search for similarity method of Pearson and Lipman
(Proc. Natl. Acad. Sci. USA 85:2444 (1988)) by computerized
implementations of algorithms GAP, BESTFIT, FASTA, and TFASTA in
the Wisconsin Genetics Software Package (Release 7.0. Genetics
Computer Group, Madison, Wis.) or by inspection. The best
alignment, (i.e. resulting in the highest percentage of homology
over the comparison window, i.e., 150 or 200 amino acids) generated
by the various methods is preferably selected. The percentage of
sequence identity is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical amino acid occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison (i.e. the window size), and multiplying the
results by 100 to yield the percentage of sequence identity.
[0054] Aequorea-related fluorescent proteins include, for example,
and without limitation, wild-type (native) Aequorea victoria GFP
(Prasher, Gene 11 1:229-233 (1992). whose nucleotide sequence (SEQ
ID NO:1) and deduced amino acid sequence (SEQ ID NO:2), allelic
variants or other variants of this sequence (for example, Q80R,
which has the glutimine residue at position 80 substituted with
arginine (Chalfie et al, Science, 263:802-805 (1994)), those
Aequorea-related engineered versions described in TABLE I, variants
that include one or more folding mutants and fragments of these
proteins that are fluorescent, such as Aequorea green fluorescent
protein form which the two amino-terminal amino acids have been
removed from the amino- or carboxy-terminus. Several of these
contain different aromatic amino acids within the central
chromophore and fluoresce at a distinctly shorter wavelength than
wild type species. For example, mutants P4 and P4-3 contain, in
addition to other mutations, the substitution Y66H. Mutants W2 and
W7 contain, in addition to other mutations, Y66W. Other mutations,
provided as non-limiting examples are listed in TABLE I. The
following six groups each contain amino acids that are conserved
substitutions for one another.
[0055] 1) Alanine (A), Serine (S), Threonine (T),
[0056] 2) Aspartic Acid (D), Glutamic Acid (E),
[0057] 3) Asparagine (N), Glutamine (O),
[0058] 4) Arginine (R), Lysine
[0059] 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V),
and
[0060] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0061] Other mutations are set forth in U.S. Pat. No. 5,625,048 to
Tsien et al. issued Apr. 29, 1997; WO 96/23810 to Tsien et al.,
published Aug. 18, 1996; PCT/US 97/12410 to Tsien et al., filed
Jul. 16, 1997; PCT/US 97/14593 to Tsien et al., filed Aug. 15,
1997; and PCT/US 96/04059 to Tsien et al., filed Mar. 20, 1996.
1TABLE I Fluorescent mutants of Aequorea green fluorescent protein
Excita- Emis- Extinction tion sion Co- Quan- Muta- Max Max
efficient tum Clone tion(s) (nm) (nm) (M-1cm-1) Yield Wild None 395
508 21,000 0.77 type (475) (7,150) P4 Y66H 383 447 13,500 0.21 P4-3
Y66H 381 445 14,000 0.38 Y145F W7 Y66W 433 475 18,000 0.67 N1461
(453) (501) (17,100) M153T V163A N212K W2 Y66W 432 480 10,000 0.72
I123V (453) (9,600) Y145H H148R M153T V163A N212K S65T S65T 489 511
39,200 0.68 P4-I S65T 504 514 14,500 0.53 M153A (396) (8,600) K238E
S65A S65A 471 504 S65C S65C 497 507 S6SL S65L 484 510 Y66F Y66F 360
442 S65T S65T 489 511 39,200 0.68 Y66H Y66H 382 448 Y66W Y66W 458
480 K4 SEQ ID 471 505 NO:.49 -2M -1G M1R S2R K3R G4A E5S E61 L71
S65A N149K V163A I1671 K5 K4 + K79N 471 505 K6 K4 + E90N 471 505 K7
K4 + E90K 471 505 K8 K4 + K79R 471 505 E90N K9 K4 + K79R 471 505
E90K K10 K4 + K79H 471 505 E90N K11 K4 + K79H 471 505 E90K K12 K4 +
K79H 471 505 K13 K4 + K79E 471 505 E90N K14 K4 + K79E 471 505 E90K
K15 K4 + K79E 471 505 K16 K4 + K79Q 471 505 K4 + E90A K4 + E90A 471
505 K4 + E90L K4 + E90L 471 505 K4 + E90V K4 + E90V 471 505 K4 +
E90T K4 + E90T 471 505 K4 + E90S K4 + E90S 471 505 K4 + E901 K4 +
E901 471 505 K4 + K79R K4 + K79R 471 505 1167T 1167T 471 502 T2031
T2031 H9 S202F 398 511 T2031 P9 I167V 471 502 (396) (507) P11 I167T
471 502 (396) (507) 10C S65G 513 527 V68L S72A T203Y W1B F64L 432
476 S65T (453) (503) Y66W N1461 M153T V163A N212K Emerald S65T 487
508 S72A N149K M153T I167T Topaz S65G 514 528 S72A K79R T203Y P4-SE
Y66H 382 446 Y145F F64L V163A Sapphire S72A 395 571 Y145F T2031
[0062] Additional mutations in Aequorea-related fluorescent
protein, referred to as "folding mutations," improve the ability of
GFP to fold at higher temperatures, and to be more fluorescent when
expressed in mammalian cells, but can have little effect on the
peak wavelengths of excitation and emission. It should be noted
that these folding mutants can be combined with mutations that
influence the spectral properties of GFP to produce proteins with
altered spectral and folding properties. Folding mutations include
the following mutations: T44A, F64L, V68L,S72A, F99S, Y145F, N1461,
S147P, M153T or A, V163A, 1167T, S175G, S205T and N212K.
[0063] This invention contemplates the use of other fluorescent
proteins in fluorescent protein substrates. The cloning and
expression of yellow fluorescent protein from Vibrio fisheri strain
YU-1 has been described by Baldwin et al. (Biochemistry
29:5509-5515 (1990)). This protein requires flavins as fluorescent
cofactors. The cloning of Peridinin chlorophill a binding protein
from the dinoflagellate Symbioclinium sp., was described by Morris
et al, (Plant Molecular Biology 24:673-677 (1994)). One useful
aspect of this protein is that is fluoresces red. The cloning of
the phycobiliprotiens from marine cyanobacteria such as
Synechoccus, e.g., phycoerythrin and phycocyanin, is described in
Wilbands et al., (J. Biol. Chem. 268:1226-12235 (1993). These
proteins sequence phycobilins as fluorescent co-factors, whose
insertion into the proteins involves auxiliary enzymes. These
protein fluoresce at yellow to red wavelengths.
[0064] It has been found that fluorescent proteins can be
genetically fused to other proteins and used as markers to identify
the location and amount of the target protein produced.
Accordingly, this invention provides fusion proteins comprising a
fluorescent protein moiety and additional amino acid sequences such
as amino acid sequences encoding a protein or polypeptide or
peptide of interest. Such additional amino acid sequences can be,
for example, up to about 15, up to about 150 or up to about 1,000
amino acids long and comprise a substrate site for an activity,
such as an enzymatic activity. The fusion proteins possess the
ability to fluorescence when excited by electromagnetic radiation.
In one embodiment, the fusion protein comprises a polyhistidine tag
to aid in purification of the protein or a poly-Lys portion to aid
in membrane association of the fluorescent compound.
[0065] Fluorescent protein substrates for a protein kinase are the
subset of fluorescent proteins as defined above whose amino acid
sequence includes a phosphorylation recognition motif and site.
Fluorescent protein substrates can be made by modifying the amino
acid sequence of an existing fluorescent protein to include a
phosphorylation recognition motif and site for a protein
kinase.
[0066] A consensus phosphorylation recognition motif for protein
kinase A is RRXSZ (SEQ ID NO:3) or RRXTZ (SEQ ID NO:4), wherein X
is any amino acid and Z is a hydrophobic amino acid, preferably
valine, leucine or isoleucine. Many variations in the above
sequence are allowed, but generally exhibit poorer kinetics. For
example lysine (K) can be substituted for the second arginine. Many
consensus sequences for other protein kinases have been tabulated
(e.g. by Kemp and Pearson, Trends Biochem. Sci. 15:342-346 (1990);
Songyang et al., Current Biology 4:973-982 (1994); Nishikawa et
al., J. Biol. Chem. 272952-960 (1997); and Songyang et al., Mol.
Cell. Biol. 16:6486-6493 (1996)).
[0067] For example, a fluorescent protein substrate selective for
phosphorylation by cGMP-dependent protein kinase can include the
following consensus phosphorylation recognition motif sequence:
BKISASEFDRPLR (SEQ ID NO:5), where B represents either lysine (K)
or arginine (R), and the first S is the site of phosphorylation
(Colbran et al, J. Biol. Chem. 267:9589-9594 (1992)). The residues
DRPLR (SEQ ID NO:6) are less important than the phenylalanine (F)
just preceding them for specific recognition by cGMP-dependent
protein kinase in preference to cAMP-dependent protein kinase.
[0068] Either synthetic or naturally occurring phosphorylation
recognition motifs can be used to create a protein kinase
phosphorylation site. For example, peptides including the motif
XRXXSXRX (SEQ ID NO:7), wherein X is any amino acid, are among the
best synthetic substrates (Kemp and Pearson, supra) for protein
kinase C. Alternatively, the Myristoylated Alanine-Rich Kinase C
substrate ("MARCKS") is one of the best substrates for PKC and is
an efficient real target for the kinase in vivo. The Examples set
forth additional substrates for PKC. The phosphorylation
recognition motif sequence around the phosphorylation site of
MARCKS is KKKKRFSFK (SEQ ID NO:8) (Graffet al., J. Biol. Chem.
266:14390-14398 (1991)). Either of these two sequences can be
incorporated into a fluorescent protein to make it a substrate for
protein kinase C.
[0069] A protein substrate for Ca.sup.2+/calmodulin-dependent
protein kinase I is derived from the sequence of synapsin, a known
optimal substrate for this kinase. The phosphorylation recognition
motif around the phosphorylation site is LRRLSDSNF (SEQ ID NO:9)
(Lee et al., Proc. Natl. Acad. Sci. USA 91:6413-6417 (1994).
[0070] A protein substrate selective for
Ca.sup.2+/calmodulin-dependent protein kinase II is derived from
the sequence of glycogen synthase, a known optimal substrate for
this kinase. The recognition sequence around the phosphorylation
site is KKLNRTLTVA (SEQ ID NO:10) (Stokoe et al. Biochem. J.
296:843-849 (1993)). A small change in this sequence to KKANRTLSVA
(SEQ ID NO: 11) makes the latter specific for MAP kinase activated
protein kinase type 1. Other preferred kinase recognition motifs
and sites are provided in TABLE II below. One skilled in the art
would realize that many proteins that do not contain such preferred
phosphorylation motifs and sites can be phosphorylated if they
conform to the consensus motif, but that the rates of
phosphorylation can be less than for the preferred substrates.
2TABLE II Underlined residue is phosphorylation site. SEQ ID NO
Protein Kinase Sequence SEQ ID NO. 50 Cyclin B-CDC2 HHHKSPRRR SEQ
ID NO. 51 Cyclin A-CDK2 HHHRSRPKR SEQ ID NO. 52 Protein Kinase A
RRRRSIIFI SEQ ID NO. 53 SLK I RRFGSLRRL SEQ ID NO. 54 ERK I
TGPLSPGPF SEQ ID NO. 55 Protein Kinase C.alpha. RRRRRKGSFRRKA
[0071] In one embodiment, the fluorescent protein substrate
contains a phophorylation motif and site at or about one or more of
the termini, in particular, the amino-terminus, of the fluorescent
protein moiety. The site preferably is located in a position within
five, ten, fifteen, or twenty amino acids of a position
corresponding to the wild type amino-terminal amino acid of the
fluorescent protein moiety. This includes sites engineered into the
existing amino acid sequence of the fluorescent protein moiety and
can also be produced by extending the amino terminus of the
fluorescent protein moiety.
[0072] One may, for example, modify the existing sequence of wild
type Aequorea GFP, or a variant thereof, to include a
phosphorylation site within the first ten, twelve, fifteen,
eighteen or twenty amino acids of the N-terminal met of wild-type
Aeqiorea GFP (or its equivalent in a fusion protein). In one
embodiment, the naturally occurring sequence is modified as
follows:
3 Wild type: MSKGEELFTG residues (1 to 10 of SEQ ID NO:2)
Substrate: MRRRRSIITG. (SEQ ID NO:12)
[0073] One may include modifying the naturally occurring sequence
of Aequorea GFP by introducing a phosphorylation motif or site into
an extended amino acid sequence of such a protein created by adding
flanking sequences to the amino terminus, for example:
4 Wild type: MSKGEELFTG residues (1 to 10 of SEQ ID NO:2) Substrate
MRRRRSIIIIFTG. (SEQ ID NO:13)
[0074] Fluorescent protein substrates having a phosphorylation site
at or about a terminus of a fluorescent protein moiety offer the
following advantages. First, it is often desirable to append
additional amino acid residues onto the fluorescent protein moiety
to create a specific phosphorylation consensus sequence. Such a
sequence is less likely to disrupt the folding pattern of a
fluorescent protein moiety when appended onto the terminus than
when inserted into the interior of the protein sequence. Second,
different phosphorylation motits can be interchanged without
significant disruption of GFP, thereby providing a general method
of measuring different kinases. Third, the phosphorylation site is
preferably exposed to the surface of the protein and, therefore,
more accessible to protein kinases. Fourth, we have discovered that
phosphorylation at sites close to the N-terminus of GFP can provide
large changes in fluorescent properties if the site of
phosphorylation is chosen such that the Ser or Thr residue that is
phosphorylated occupies a position that was originally negative or
positively charged in the wild-type protein. Specifically.
replacement of Glu 5 or Glu 6 by a non-charged Ser or Thr residue
can significantly disrupt the fluorescence, folding, or sensitivity
of GFP to quenching. Phosphorylation of the serine or threonine can
restore negative charge to this position and thereby increases the
ability of GFP to fold correctly at higher temperatures, and hence
can increase the fluorescence of GFP and resistance to
quenching.
[0075] In another embodiment, the fluorescent protein substrate
includes a phosphorylation site remote form a terminus, e.g., that
is separated by more than about twenty amino acids from the
terminus of the florescent protein moiety and within the
fluorescent protein moiety. One embodiment of this form includes
the Aequorea-related fluorescent protein substrate comprising the
substitution H217S, creating a consensus protein kinase A
phosphorylation motif and site. Additionally, phosphorylation
recognition motifs comprising the following alterations based on
the sequence of wild type Aequorea GFP exhibit fluorescent changes
upon phosphorylation: 69RRFSA (SEQ ID NO: 14) and 214KRDSM (SEQ ID
NO:15) (which includes H217S).
[0076] The artisan should consider the following when selecting
amino acids for substitution within the fluorescent protein moiety
remote in primary amino acid sequence form the terminus. First, it
is preferable to select amino acid sequences within the fluorescent
protein moiety that resemble the sequence of the phosphorylation
motif and site. In this way, fewer amino acid substitutions in the
native protein are needed to introduce the phosphorylation motif
and site into the fluorescent protein. For example, protein kinase
A recognizes the sequence RRXSZ (SEQ ID NO:3) or RRXTZ (SEQ ID
NO:4), wherein X is any amino acid and Z is a hydrophobic amino
acid. Ser or Thr is the site of phosphorylation. It is preferable
to introduce this sequence into the fluorescent protein moiety at
sequences already containing Ser or Thr so that Ser or Thr are not
substituted in the protein. More preferably, the phosphorylation
recognition motif is created at locations having some existing
homology to the sequence recognized by protein kinase A, e.g.
having a proximal Arg or hydrophobic residues with the same spatial
relationship as in the phosphorylation site.
[0077] Second, location on the surface of the fluorescent protein
is preferred for phosphorylation sites. This is because surface
locations are more likely to be accessible to protein kinaes than
interior locations. Surface locations can be identified by computer
modeling of the fluorescent protein structure or by reference to
the crystal structure of Aequorea GFP. Also, charged amino acids or
groups of charged amino acids in the fluorescent protein are more
likely to lie on the surface than inside the fluorescent protein,
because such amino acids are more likely to be exposed to water in
the environment.
[0078] In cases where the phosphorylation site is either at a
terminus, such as the N-terminus, or remote from a terminus, the
amino acid context around the phosphorylation site can be optimized
in order to maximize the change in fluorescence. Amino acid
substitutions that change large bulky and/or hydrophobic amino
acids to smaller and less hydrophobic replacements are generally
helpful. Similarly, large charged amino acids can be replaced by
smaller, less charged amino acids. For example:
[0079] a) Hydrophobic to less hydrophobic
[0080] Phe to Leu
[0081] Leu to Ala
[0082] b) Charged to charged but smaller
[0083] Glu to Asp
[0084] Arg to Lys
[0085] c) Charged to less charged
[0086] Glu to Gln
[0087] Asp to Asn
[0088] d) Charged to polar
[0089] Glu to Thr
[0090] Asp to Ser
[0091] e) Changed to non-polar
[0092] Glu to Leu
[0093] Asp to Ala
[0094] These changes can be accomplished by direct means or using
random iterative approaches where changes are made randomly and the
best ones selected based upon their change in fluorescent
properties after phosphorylation by an appropriate kinase.
[0095] Third, amino acids at distant locations from the actual site
of phosphorylation can be varied to enhance fluorescence changes
upon phosphorylation. These mutations can be created through site
directed mutagenesis, or through random mutagenesis, for example by
error-prone PCR, to identify mutations that enhance either absolute
fluorescence or the change in fluorescence upon phosphorylation.
The identification of mutants remote in primary sequence from a
terminus, such as an N-terminus, identifies potentially interacting
sequences that may provide additional areas in which further
mutagenesis can be used to refine the change in fluorescence upon
phosphorylation. For example, mutations around the amino terminus
phosphorylation site may interact (either transiently during
folding, or in a stable fashion) with amino acids at positions 171
and 172, and point mutations that significantly disrupt
fluorescence of GFP by changing negative to positive charges near
the amino terminus can be rescued by changing a positive to a
negative charge at position 171.
[0096] In the phosphorylation mutant below the sequence is a), the
wild type sequence b) is also listed below.
5 a) MSKRRDSLT (SEQ ID NO:16) b) MSKGEELFT (1 to 9 of SEQ ID
NO:2)
[0097] The phosphorylation mutant has only 7% of the fluorescence
of the wild type protein. However, its fluorescence can be restored
to 80% of the wild type by two amino acid changes, E171K and I172V,
positions which are quite remote in linear sequence form the amino
terminus.
[0098] Thus, changes in charge at E171 K (negative to positive) can
almost completely restore the fluorescence of the phosphorylation
mutant, strongly suggesting that the original loss of fluorescence
arose primarily through changes in charge caused by the point
mutations. It is clear that the addition and loss of charge at
positions around, and at the phosphorylation site, have a
significant impact on fluorescence formation. The fact that charge
alone can significantly affect the fluorescence properties of GFP
is highly significant within the scope of the present application
since phosphorylation involves the addition of two negative changes
associated with the phosphate group on the serine residue.
[0099] In the above case the mutations restore fluorescence of the
phosphorylation mutant without significantly increasing the
magnitude of the change in fluorescence upon phosphorylation.
Nevertheless, the identification of these positions in GFP provides
a valuable tool to further enhance changes in fluorescence upon
phosphorylation by creating random mutations at codons around
positions 171, 172, and 173 to identify mutations that enhance
changes in fluorescence upon phosphorylation. This can be achieved
by co-expressing the kinase of interest with the fluorescent
substrate of the present invention containing random mutations that
may enhance the fluorescence changes upon phosphorylation.
[0100] A GFP-based phosphorylation sensor having a phosphorylation
motif or site at or near the amino-terminus can be modified to
establish a phosphorylation sensor having enhanced fluorescence,
enhanced kinetics of phosphorylation, and enhanced changes in
fluorescence upon quenching. Within the amino terminal sequence of
GFP the first four amino acids are freely interchangeable. The next
seven amino acids can be modified, preferably with conservative
amino acid changes, to accommodate a phosphorylation recognition
motif. To achieve high levels of fluorescence, it may be preferable
to mutate addition sites in GFP to promote efficient folding. These
methods are discussed in the Examples. In addition to enhancing
fluorescence of such sensors, the kinetics of phosphorylation of
these sensors can be enhanced. For example, the efficiency with
which a phosphorylation site is modified by a kinase or phosphatase
is dependent on the sequence and accessibility of the recognition
motif. The accessibility of the phospohrylation motif can be
improved my making changes in amino acids that disorder the local
amino-terminal structure of GFP or reduce interactions between the
amino-terminal region and the interior of the molecule, preferably
by disrupting interactions between Lys3 and Glu90 and amino acids
around these residues.
[0101] Preferable mutants can be identified using, for example, the
following method. Nucleic acid molecules encoding such fluorescent
compounds, such as kinase sensors, can be placed into an
appropriate expression vector. The expression vector can also
encode an activity, such as an activity specific for the
fluorescent compound, such as a kinase. The expression vectors are
transformed into host cells, such as bacteria or mammalian cells,
such as human cells, and the individual bacterial colonies grown
up. to Each colony is derived from a single cell, and hence
contains a single unique mutant fluorescent substrate grown up. The
individual colonies may then be grown up and screened for
fluorescence either by fluorescence activated cell sorting (FACS),
or by observation under a microscope. Those that exhibit the
greatest fluorescence can then be screened under conditions in
which the gene encoding the activity, such as a kinase activity, is
inactivated. Appropriate digests of the kinase gene can achieve
this by restriction enzymes that specifically cut within the kinase
but not GFP. Comparison of the brightness of the mutant first in
the presence of kinase then in its absence indicates the relative
effect of phosphorylation of the mutant GFP.
[0102] Fluorescent protein substrates for a protease can be made
using the methods and strategies discussed above for fluorescent
protein substrates for a protein kinase. The skilled artisan need
merely incorporate a protease cleavage recognition site into the
fluorescent compound rather than a substrate site for a protein
kinase. Such protease cleavage recognition sites are known in the
art, some of which are presented in TABLE III.
6TABLE III Protease Sequence HIV-1 protease SQNY-PIVQ (SEQ ID NO:
37) KARVL-AEMS (SEQ ID NO: 38) Prohormone convertase PSPREGKR-SY
(SEQ ID NO: 39) Interleukin-1b-converting enzyme YVAD-G (SEQ ID
NO.: 40) Adenovirus endopeptidase MFGG-AKKR (SEQ ID NO: 41)
Cytomegalovirus assemblin GVVMA-SSRLA (SEQ ID NO: 42)
Leishmanolysin LIAYI-LKKAT (SEQ ID NO: 43) b-Secretase for amyloid
precursor protein VKM-DAEF (SEQ ID NO: 44) Thrombin
FLAEGGGVR-GPRVVERH (SEQ ID NO: 45) DRVYIHPF-HL-VIH (SEQ ID NO. 46)
Renin and angiotensin-converting Enzyme Cathepsin D KPALF-FRL (SEQ
ID NO: 47) Kininogenases including kallikrein QPLGQTSLMK-RPPGFSPER
SVQVMKT QEGS (SEQ ID NO: 48)
[0103] See, e.g., Matayoshi et al. (1990) Science 247:954, Dunn et
al. (1994) Meth. Enzymol. 241:254, Seidah I & Chretien (1994)
Meth. Enzymol. 244:175, Thomberry (1994) Meth. Enzymol. 244:615,
Weber & Tihanyi (1994) Meth. Enzymol. 244:595, Smith et
al.(1994) Meth. Enzymol. 244:412, Bouvier et al. (1995) Meth.
Enzymol. 248:614, Hardyi et al. (1994) in Amyloid Protein Precursor
in Development, Aging, and Alzheimer's Disease,
[0104] The methods discussed above can be used to confirm that a
fluorescent compound comprising a protease cleavage motif and site
exhibits at least one different fluorescent property in the cleaved
and uncleaved state In addition to protein kinase substrates,
protein phosphatase substrate, and protease substrates, the present
invention encompasses substrates for protein prenyltransferases,
glycosylases, any other enzyme recited in this application, any
other known enzyme, or any enzyme later discovered. Fluorescent
compounds that are substrates for these enzyme activities can be
made using the methods described in the present application
following the exemplary teachings set forth in the Examples. For
example, different substrate recognition motifs and substrate sites
for activities, such as enzyme activities, can be incorporated into
GFP to make and confirm that compounds that can exhibit quenching
in response to an activity. Furthermore, fluorescent compounds
other than GFP that have at least one substrate recognition motif
and site for activities can be made and confirmed to exhibit
quenching in response to an activity by following the exemplary
teachings set forth in the Examples.
[0105] Furthermore, the present invention encompasses substrate
sites for an activity, such as an enzymatic activity, that exhibits
at least one different fluorescent property in a first and second
state after quenching that can be detected by fluorescent detection
methods. Such activities include proteases, transferases,
glycosylases, reductases, oxidases, or any other enzyme recited in
this application, any other known enzyme, or any enzyme later
discovered. Such substrates can be made using the exemplary
teachings set forth in the Examples.
[0106] Nucleic Acid Molecules Coding for the Expression of a
Fluorescent Compound
[0107] While certain florescent compounds can be prepared
chemically, for example, by coupling a fluorescent moiety to the
amino terminus of a protein moiety, it is preferable to produce
fluorescent compounds comprising a peptide or protein
recombinantly.
[0108] Recombinant production of a fluorescent compound involves
expressing a nucleic acid molecule having sequences that encode a
peptide or protein. As used herein, the term "nucleic acid
molecule" includes both DNA and RNA molecules. It will be
understood that when a nucleic acid molecule is said to have a DNA
sequence, this also includes RNA molecules having the corresponding
RNA sequence in which "U" replaces "T." The term "recombinant
nucleic acid molecule" refers to a nucleic acid molecule which is
not naturally occurring, and which comprises two nucleotide
sequences that are not naturally joined together. Recombinant
nucleic acid molecules are produced by artificial combination,
e.g., genetic engineering techniques or chemical synthesis.
[0109] In one embodiment, the nucleic acid encodes a fusion protein
in which a single polypeptide includes the fluorescent protein
moiety within a longer polypeptide In another embodiment, the
nucleic acid encodes the amino acid sequence that comprises a
substrate site for an activity consisting essentially of a
fluorescent protein moiety modified to include a substrate site for
an activity. In either case, nucleic acids that encode fluorescent
proteins are useful as starting materials.
[0110] Nucleic acids encoding a fluorescent protein moiety can be
obtained by methods known in the art. For example, a nucleic acid
encoding a GFP can be isolated by polymerase chain reaction of cDNA
from A. victoria using primers based on the DNA sequence of A.
victoria green fluorescent protein (SEQ ID NO:2). PCR methods are
described in, for example, U.S. Pat. No. 4,683,195; Mullis et al,
cold Spring Harbor Symp. Quant. Biol. 51:263 (1987). and Erlich,
PCR Technology, Stockton Press, NY (1989).
[0111] Mutant versions of fluorescent proteins can be made by
site-specific mutagenesis of other nucleic acids encoding a
fluorescent protein moiety or by random mutagenesis caused by
increasing the error rate of PCR of the original polynucleotide
with 0.1 mM MnCl.sub.2 and unbalanced nucleotide concentrations
(U.S. Pat. No. 5,625,048 to Tsien, issued Apr. 29, 1997; and
PCT/US95/14692, filed Nov. 10, 1995).
[0112] Nucleic acids encoding fluorescent compounds that are
fusions between a polypeptide including a phosphorylation site and
a fluorescent protein moiety can be made by ligating nucleic acids
that encode each of these. Nucleic acids encoding fluorescent
compounds that include the amino acid sequence of a fluorescent
protein moiety in which one or more amino acids in the amino acid
sequence of a fluorescent protein moiety are substituted to create
a substrate site for an activity can be created by, for example,
site specific mutagenesis of a nucleic acid encoding a fluorescent
protein moiety.
[0113] Nucleic acids used to transfect cells with sequences coding
for expression of a polypeptide of interest such as those encoding
a fluorescent compound generally will be in the form of an
expression vector including expression control sequences
operatively linked to a nucleotide sequence coding for expression
of the polypeptide. As used herein, the term "nucleotide sequence
coding for expression of a polypeptide" refers to a sequence that,
upon transcription and translation of mRNA, produces the
polypeptide. As any person skilled in the art recognizes, this
includes all degenerate nucleic acid sequences encoding the same
amino acid sequence. This can include sequences containing, e.g.
introns. As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are "operatively linked" to a nucleic acid
sequence when the expression control sequence control and regulate
the transcription and, as appropriate, translation of the nucleic
acid sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription termination, or a
start codon. (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
stop codons. Recombinant nucleic acid can be incorporated into an
expression vector comprising expression control sequences
operatively linked to the recombinant eukaryotes by inclusion of
appropriate promoters, replication sequences. market, etc. The
expression vector can be transfected into a host cell for
expression of the recombinant nucleic acid. Host cells can be
selected for high levels of expression in order to purify the
protein. E. coli is useful for this purpose. Alternatively, the
host cell can be a prokaryotic or eukaryotic cell selected to study
the activity of an enzyme produced by the cell. The cell can be,
e.g. a cultured cell or a cell in vivo. The construction of
expression vectors and the expression of genes in transfected cells
involves the use of molecular cloning techniques also well known in
the art (Sambrook et al., Molecular Cooing--A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989);
Ausubel et la., Current Protocols in Molecular Biology, Greene
Publishing Associates and John Wiley & Sons, Inc.).
[0114] Recombinant fluorescent protein substrates can be produced
by expression of nucleic acid encoding for the protein in E. coli.
Aequorea-related florescent proteins are best expressed by cells
cultured between about 15.degree. C. and 30.degree. C. but higher
temperatures (e.g. 37.degree. C.) are possible. After synthesis,
these enzymes are stable at higher temperatures (e.g. 37.degree.
C.) and can be used in assay at those temperatures.
[0115] The construct can also contain a tag to simplify isolation
of the expressed fluorescent compound. For example, a polyistidine
tag of, e.g. six histidine residues, can be incorporated at the
amino or carboxyl terminal of the fluorescent compound. The
polyhistidine tag allows convenient isolation of the protein in a
single step by nickel chromatography.
[0116] Alternatively, the fluorescent compound, such as a
fluorescent protein substrate, need not be isolated from the host
cells. This method is particularly advantageous for the assaying
for the presence of an activity in situ.
[0117] Methods for Determining Whether a Sample Contains an
Activity
[0118] The present invention includes methods for determining
whether a sample contains an activity using a fluorescent compound
of the present invention. Depending on the type of activity to be
determined, different fluorescent compounds are to be used. For
example, if a protease activity is to be determined, then a
fluorescent compound that is a substrate for a protease is used in
the present methods. Likewise, if a protein kinase activity is to
be determined, then a fluorescent compound that is a substrate for
a protein kinase is used in the present invention.
[0119] The present method for determining whether a sample contains
an activity comprises contacting a sample with a fluorescent
compound of the present invention, wherein said fluorescent
compound exhibits quenching. The sample is contacted with a
quenching agent, excited, and the amount of emission from the
fluorescent compound under quenching conditions is measured.
[0120] As is known in the art, different cofactors are required for
different enzyme reactions. Thus, the skilled artisan would realize
that such cofactors should be present in the assay conditions for
those enzymes. For example, protein kinases add a phosphate residue
to the phosphorylation site of a protein generally through the
hydrolysis of ATP to ADP. Fluorescent compounds that are substrates
for protein kinases are useful in assays to determine the amount of
protein kinase activity in a sample. The assays of this invention
take advantage of the fact that phosphorylation of the protein
results in a change in a fluorescent property of the fluorescent
compound that can be detected by quenching. Methods for determining
whether a sample has kinase activity involve contacting the sample
with a fluorescent compound having a phosphorylation site
recognized by the protein kinase to be assayed and with a phosphate
donor under selected test conditions. A phosphate donor is a
compound containing a phosphate moiety that the kinase is able to
use to phosphorylate the protein substrate. ATP
(adenosine-5'-triphosphat- e) is by far the most common phosphate
donor. In certain instances (such as whole cell studies) the sample
will contain enough of a phosphate donor to make this step
unnecessary. Then the fluorescent compound is excited with light in
its excitation spectrum in the presence and absence of at least one
quenching agent. If the fluorescent compound has been
phosphorylated, the substrate will exhibit resistance to quenching,
indicating that the sample contains protein kinase activity. These
general methods can be used to detect any activity using
fluorescent compound, assay conditions, and quenching conditions
appropriate for an activity.
[0121] The amount of an activity in a sample can be determined by
measuring the amount of quenching in the sample at a first time and
a second time after contact between the sample and the fluorescent
protein compound and determining the degree of change or the rate
of change in a quenching. These results can be compared to those
obtained with a control sample that does not contain the activity,
or contains a known amount of activity. The amount of an activity
in the sample can be calculated as a function of the difference in
the determined amount of quenching at the two times. For example,
the absolute amount of an activity can be calibrated using
standards of activity determined for certain amounts of activity
after certain amounts of time. The faster or larger the difference
in the amount of quenching, the more activity is present in the
sample. The skilled artisan would realize that proper controls
should be used to validate any comparisons made with data obtained
from the sample.
[0122] Fluorescence in a sample is measured using a fluorimeter. In
general, excitation radiation from an excitation source having a
first wavelength, passes through excitation optics. The excitation
optics causes the excitation radiation to excite the sample. In
response, fluorescent compounds in the sample emit radiation that
has a wavelength that is different from the excitation wavelength.
Collection optics then collect the emission from the sample. The
device can include a temperature controller to maintain the sample
at a specific temperature while it is being scanned. According to
one embodiment, a multi-axis translation stage moves a microtiter
plate holding a plurality of samples in order to position different
wells to be exposed. The multi-axis translation stage, temperature
controller, auto-focusing feature, and electronics associated with
imaging and data collection can be managed by an appropriately
programmed digital computer. The computer also can transform the
data collected during the assay into another format for
presentation. This process can be miniaturized and automated to
enable screening many thousand of compounds.
[0123] For example, comparisons can be made with a control sample
known not to contain an activity, a control sample known to contain
an activity (preferably in a known amount), a control sample
representing background signal, or a control sample with or without
test compounds.
[0124] The sample can be any sample, such as a sample of cells,
tissue, organ, or fluid obtained from an organism (such as a
mammalian, such as a human) or an extract obtained therefrom.
Miniaturized arrays of samples attached to a matrix, such as a bead
or solid support as they are known in the art or later developed,
can be used in the present invention to detect fluorescence or
other activity in a sample. A sample can also comprise cultured
cells, culture fluid, or extracts or conditioned media obtained
therefrom. The cells can be prokaryotic or eukaryotic, such as
mammalian cells, such as human cells.
[0125] Methods of performing assays on fluorescent materials are
well known in the art. (Lakowics, Principles of Fluorescence
Spectroscopy, Plenum Press, NY (1983); Herman, Fluorescence
Microscopy of Living Cells in Culture, Part B, Methods in Cell
Biology, volume 30, Academic Press, San Diego, pp. 219-243 (1989);
Turro, Modern Molecular Photochemistry, Menlo Park, Calif.,
Benjamin/Cummings Publishing, pp. 296-361 (1978)).
[0126] In one embodiment, a cell is transiently or stably
transfected with an expression vector encoding a fluorescent
compound containing a substrate site for an activity to be assayed.
This expression vector optionally includes controlling nucleotide
sequences such as promoter or enhancing elements. The expression
vector expresses the fluorescent compound that contains the
substrate site for an activity to be detected. The activity to be
assayed may either be intrinsic to the cell or may be introduced by
stable transfection or transient co-transfection with another
expression vector encoding the activity and optionally including
controlling nucleotide sequences such as promoter or enhancer
elements. The fluorescent compound and the activity preferably are
located in the same cellular compartment so that they have more
opportunity to come into contact. Membrane-bound or
membrane-associated fluorescent compounds can also be used in this
and any other method of the present invention. The amount of
activity is then determined by measuring the fluorescence of the
sample (which can contain whole cells) under quenching conditions,
and comparison to appropriate controls, such as controls that
either do not contain the activity, or contain a known amount of
activity.
[0127] A contemplated variation of the above assay is to use the
controlling nucleotide sequence to produce a sudden increase in the
expression of either the fluorescent compound or the enzyme being
assayed, for example, by inducing expression of the construct.
Fluorescence after quenching can be monitored at one or more time
intervals after the onset of increased expression. A large
difference in the amount of fluorescence after quenching over time
reflects a large amount or high efficiency of the activity. This
kinetic determination has the advantage of minimizing any
dependency of the assay on the basal or background levels of
activity.
[0128] In another embodiment, the vector may be incorporated into
an entire organism by standard transgenic or gene replacement
techniques. An expression vector capable of expressing an activity
optionally may be incorporated into the entire organism by standard
transgenic or gene replacement techniques. Then, a sample from the
organism containing the fluorescent compound is tested. For
example, cell or tissue homogenates, individual cells, or samples
of body fluids, such as blood, can be tested.
[0129] Screening Assays
[0130] The methods of the invention can be used in drug screening
to determine whether a test compound alters an activity. In one
embodiment, the assay is performed on a sample in vitro suspected
of containing an activity. A sample containing a known amount of
activity is mixed with a fluorescent compound of the invention and
a test compound. The amount of the activity in the sample is then
determined as above, for example by measuring the amount of
fluorescence after quenching at a first and optionally second time
after contact between the sample, the fluorescent compound, and the
test compound and at least one quenching agent. Then the amount of
activity in the presence of the test compound is compared with the
activity in the absence of the test compound. A difference
indicates that the test compound alters the activity. The activity
can be increased, decreased, or unchanged by a test compound.
[0131] The present invention also includes a compound identified by
any method of the present invention. Such compounds can be provided
as a pharmaceutical composition in a pharmaceutically acceptable
carrier as is set forth in U.S. patent application Ser. No.
09/030,578, filed Feb. 24, 1998. The present invention also
includes a library of such compounds, which comprise two or more of
such compounds provided either separately or in combination. The
present invention also includes a system used to screen and
identify compounds, such as set forth in U.S. patent application
Ser. No. 08/858,016, filed May 16, 1997.
[0132] In another embodiment, the ability of a compound to alter an
activity in vivo is determined. In an in vivo assay, cells
transfected with an expression vector encoding a fluorescent
compound, such as a fluorescent protein, of the invention are
exposed to at least one amount of at least one test compound, and
the fluorescence after quenching in each cell (individually or as a
population) can be determined. Typically, the difference is
calibrated against standard measurements (for example, in the
presence or absence of test compounds) to yield an absolute amount
of activity. A test compound that inhibits or blocks the activity
or expression of an activity can be detected by a relative change
in fluorescence after quenching. The cell can also be tranfected
with an expression vector to coexpress the activity or an upstream
signaling component such as a receptor, and the fluorescent
compound. This method is useful for detecting signaling to an
activity such as a protein kinase of interest from an upstream
component of a signaling pathway. If a signal from an upstream
molecule, for example a receptor (preferably in the presence of an
agonist), is inhibited by a test compound, then the kinase activity
will be inhibited as compared to controls incubated without the
test compound. This provides a method for screening for compounds
that affect cellular events (including receptor-ligand binding,
protein-protein interaction, or kinase activation), and signal to
the target kinase. This method can use cultured cells or extracts
or conditioned media derived therefrom. This method can also use
cells derived from an organism, such as a mammal, such as a human.
Such cells can be derived from a tissue, organ or fluid. The sample
can also comprise an extract of such cells.
[0133] This invention also provides kits containing a fluorescent
compound and optionally cofactors for an activity. In one
embodiment, the kit comprises at least one container holding the
fluorescent compound and optionally a second container holding a
cofactor or buffer. Optionally, the kit can comprise other reagents
or labware to practice a method, such as a method of the present
invention. The entire kit can be provided in a separate container,
such as a box. This container can include instructions for use of
the contents in a method of the present invention, or for other
purposes.
[0134] Libraries of Candidate Substrates
[0135] This invention provides libraries of fluorescent compounds
useful for the identification and characterization of sequences
that can be recognized by an activity. Libraries of these
fluorescent compounds can be screened to identify sequences that
can be modified by activities of unknown or known substrate
specificity, or to characterize differences in activity in, or
from, diseased and normal cells, tissues, fluids, or extracts
thereof.
[0136] As used herein, a "library" refers to a collection
containing at least two different members, preferably greater than
ten different members. Each member of a fluorescent compound
library comprises a fluorescent protein moiety and a variable
substrate site for an activity, wherein the variable substrate site
for an activity is preferably located at or near the amino- or
carboxy-terminus of the fluorescent protein moiety and has fewer
than about fifty amino acids, preferably less than about fifteen
amino acids. The variety of amino acid sequences for the
fluorescent protein moiety is at the discretion of the artisan. For
example, the library can contain a diverse collection of variable
peptide moieties in which most or all of the amino acid positions
are subjected to a non-zero but low probability of substitution.
Also, the library can contain variable peptide moieties having an
amino acid sequence in which only a few (e.g. one to ten) amino
acid positions are varied, but the probability of substitution at
each positions is relatively high.
[0137] Preferably, libraries of fluorescent compounds are created
by expressing protein from libraries of recombinant nucleic acid
molecules having expression control sequences operatively linked to
nucleic acid sequences that code for the expression of different
fluorescent compounds. Methods of making nucleic acid molecules
encoding a diverse collection of peptides are described (U.S. Pat.
No. 5,432,018 to Dower et al., U.S. Pat. No. 5,223,4098 to Ladner
et al., U.S. Pat. No. 5,264,563 to Huse et al., and WO 92/06176 to
Huse et al.) For expression of fluorescent compounds, recombinant
nucleic acid molecules are used to tranfect cells, such that each
cell contains a member of the library. This produces, in turn, a
library of host cells capable of expressing the library of
different fluorescent compounds. The library of host cells can be
used in the screening methods of this invention to identify
fluorescent compounds comprising a substrate site for an
activity.
[0138] In one method of creating such a library, a diverse
collection of oligonucleotides having preferably random codon
sequences are combined to create polynucleotides encoding peptides
having a desired number of amino acids. The oligonucleotides
preferably are prepared by chemical synthesis. The polynucleotides
encoding the variable peptide moieties can be coupled to the 5' end
of a nucleic acid coding for the expression of a fluorescent
compound or a carboxy-or amino-terminal portion of it. This is, the
fluorescent protein moiety or a carboxy-terminal portion of it.
This creates a recombinant nucleic acid molecule coding for the
expression of a fluorescent compound having a peptide moiety fused
to the amino- or carboxy-terminus of a fluorescent protein moiety.
This recombinant nucleic acid molecule is then inserted into an
expression vector to create a recombinant nucleic acid molecule
comprising expression control sequences operatively linked to the
sequence encoding the candidate substrate. The expression vector
can then be inserted into an appropriate cell and expressed. To
generate the collection of oligonucleotides which forms a series of
codons encoding a random collection of amino acids and which is
ultimately cloned into the vector, a codon motif is used, such as
(NNK)x, where N may be A,C,G, or T (nominally equimolar, K is G or
T (nominally equimolar), and x is the desired number of amino acids
in the peptide moiety, e.g. 15 to produce a library of 1 5-mer
peptides. The third positions may also be G or C, designated "S."
Thus, NNK or NNS (i) code for all the amino acids, (ii) code for
only one stop codon, and (iii) reduce the range of codon bias from
6:1 to 3:1. The expression of peptides from randomly generated
mixtures of oligonucleotides in appropriate recombinant vectors is
discussed in Olipant et al., Gene 44:177-183 (1986).
[0139] An exemplified codon motif (NNK)6 (SEQ ID NO:17) produces 32
codons, one for each of 12 amino acids, two for each of five amino
acids, three for each of three amino acids and one (amber) stop
codon. Although this motif produces a codon distribution as
equitable as available with standard methods of oligonucleotide
synthesis, it results in a bias against peptides containing
one-codon residues.
[0140] An alternative approach to minimize the bias against
one-codon residues involves the synthesis of 20 activated
tri-nucleotides, each representing the codon for one of the 20
genetically encoded amino acids. These are synthesized by
conventional means, removed from the support but maintaining the
base and 5-OH-protecing groups, and activating by the addition of
3'O-phosphoramidite (and phosphate protection with beta-cyanoethyl
groups) by the method used for the activation of mononucleosides,
as generally described in McBride and Caruthers, Tetrahedron
Letters 22:245 (1983). Degenerate "oligocodons" are prepared using
these trimers and building blocks. The trimers are mixed at the
desired molar rations and installed in the synthesizer. The ratios
will usually be approximately equimolar, but may be controlled
unequal ratios to obtain the over- to under-representation of
certain amino acids coded for by the degenerate oligonucleotide
collection. The condensation of the trimers to form the oligocodons
is done essentially as described for conventional synthesis
employing activated mononucleosides as building blocks (Atkinson
and Smith, Oligonucleotide Synthesis, Gait ed. pp. 35-82 (1984).
Thus, this procedure generates a population of oligonucleotides for
cloning that is capable of encoding an equal distribution (or a
controlled unequal distribution) of the possible peptide
sequences.
[0141] Methods for Screening for Quenching Agents
[0142] The present invention includes methods for screening for
agents that are quenching agents for a fluorescent compound of the
present invention. As set forth in the Examples, fluorescent
compounds that have a substrate site for an activity can exhibit
quenching in the presence of a quenching agent. Preferable
quenching agents and quenching conditions for a particular
fluorescent compound can be followed by screening a plurality of
quenching agents and quenching conditions following the methods set
forth in the Examples. For example, a first sample of a fluorescent
compound can be contacted with a control buffer and a second sample
of a fluorescent compound can be contacted with an activity. These
samples can be incubated for any period of time, such as between
about one minute and 72 hours, preferably between 1 and 6 hours.
Aliquots of these samples can then be contacted with test quenching
agents and/or test quenching conditions, after which the
fluorescence of these samples are measured. Samples that exhibit
quenching can be readily identified by measuring the appropriate
fluorescence from the samples (preferably by comparison with an
appropriate control) which identify preferable quenching agents or
quenching conditions. More preferable quenching agents or quenching
conditions can be identified by reiterating this procedure using
different concentrations of identified quenching agents for longer
or shorter periods of time.
[0143] Methods for Screening Libraries of Candidate Substrates.
[0144] Libraries of host cells expressing fluorescent compounds are
useful in identifying fluorescent proteins having peptide moieties
that exhibit quenching. Several methods of using the libraries are
envisioned. In general, one begins with a library of recombinant
host cells, each of which expresses a different fluorescent
compound, such as fluorescent compound comprising a protein,
peptide, or nucleic acid. Each cell is expanded into a clonal
population that is genetically homogeneous.
[0145] In a first method, fluorescence quenching is measured or
compared from each clonal population before and after at least one
specified time after a known change in an intracellular activity.
Alternatively, fluorescence quenching measured in each clonal
population can be compared with the results obtained using
untreated control cells. For example, a change in kinase activity
could be produced by transfection with a gene encoding a kinase
activity, by increasing the expression of the kinase using
expression control elements, or by any condition that
post-translationally modulates the kinase activity. Examples of the
latter include cell surface receptor mediated elevation of
intracellular cAMP to activate cAMP-dependent kinases, surface
receptor mediated increases of intracellular cGMP to activate
cGMP-dependent protein kinase, increases in cytosolic free calcium
to activate Ca.sup.2+/calmodulin-dependent protein kinase types I,
II, or IV, or the production of diacylglycerol to activate protein
kinase C, etc. One then selects for the clone(s) that show the
largest or fastest changes in fluorescence in response to quenching
compared to non-treated control cells.
EXAMPLES
[0146] A. Phosphorylation Sites Located in the Amino Acid Sequence
of Aequorea GFP Remote in the Primary Amino Acid Sequence Form the
N-Terminus
[0147] Potential sites for phosphorylation were chosen at or close
to positions in GFP that had previously been identified on the
basis of mutagenesis experiments to exert significant effects on
fluorescence, or which had a higher probability of surface exposure
based on computer algorithms. For example, in mutant H9, Ser 202
and Thr 203 are mutated to Phe and lie, respectively, creating a
large change in spectral properties. Therefore, in one mutant. 199
RRLSI (SEQ ID NO:18), a potential site of phosphorylation was
created around Ser 202, whose phosphorylation would significantly
affect the fluorescent properties of the parent molecule.
Similarly, the amino acids located at positions72 and 175 have been
implicated in increased folding efficiency of GFP at higher
temperatures and were made into potential sites of phosphorylation
in separate mutants.
[0148] A complete list of the positions and amino acid changes made
for each phosphorylation mutant in this series is outlined in FIG.
2. Proteins were expressed in E. coli using the expression plasmid
pRSET (Invitrogen, CA), in which the regions encoding GFP was fused
in frame with nucleotides encoding an N-terminal polyhistidine tag
(FIG. 3). The sequence changes were introduced by site-directed
mutagenesis using the Bio-Rad mutagenesis kit (Kunkel, Proc. Natl.
Acad. Sci. 82:488-492 (1985)); and Kunkel et al., Meth. Enzymol.
154:367-382 (1987)) and confirmed by sequencing. The recombinant
proteins were induced with 0.05 mM IPTG, expressed in bacteria and
purified by nickel affinity chromatography. The sequence changes,
relative fluorescence, relative rater of phosphorylation and the
percent change in fluorescence upon phosphorylation are listed in
Table IV. In those cases where the protein exhibited no
fluorescence after insertion of the phosphorylation site, no
determinations were made on the effect of phosphorylation on
fluorescence.
7TABLE IV Relative fluorescence, rate of phosphorylation and change
in fluorescence upon phosphorylation for mutants incorporating
phosphorylation sites remote from the N- terminus. % change in
Fluorescence before fluorescence after phosphorylation (% Relative
rates of incubation with SEQ ID NO: Sequence of wild type)
physphorylation kinase SEQ ID NO.19 25RRFSV 95 1.75 -5 SEQ ID NO.20
68RRFSR 0 N.D. N.D. SEQ ID NO.14 68RRFSA 6 0.6 10 SEQ ID NO.21
94RRSIF 0 N.D. N.D. SEQ ID NO.22 131RRGSIL 0 N.D. N.D. SEQ ID NO.23
155KRKSGI 86 2.5 0 SEQ ID NO.24 172RRGSV 90 1.57 0 SEQ ID NO.18
199RRLSI 0 N.D. N.D. SEQ ID NO.15 214KRDSM 21 1.88 40 N.D. means
"not determined"
[0149] Numbers prior to the sequence indicate amino acid position
in wild type GFP (SEQ ID NO:2) where phosphorylation motif starts.
The relative rates of phosphorylation compare the rate of
phosphorylation of the given phosphorylation motif with the
endogenous protein kinase A phosphorylation motif in Aequorea GFP
(HKFSV. SEQ ID NO:1) measured by incorporation of .sup.32P after
incubation of the purified substrate and protein kinase A catalytic
subunit in the presence of .sup.32P-labeled ATP using 3 micrograms
of GFP, 5 micrograms protein kinase A catalytic subunit, for 10
minutes at 30.degree. C. in standard phosphorylation buffer (20 mM
MOPS, pH 6.5, 100 mM KCl, 100 micromolar ATP, 3 mM MgC.sub.2, 1 mM
DTT and 100 microCi .sup.32P-labeled ATP. Reactions were terminated
by blotting onto phosphocellulose paper and washing with 10%
phosphoric acid. The percent change in fluorescence represents the
increase in fluorescence (475 nm excitation, 510 nm emission)
observed in each purified protein resulting from incubation with
excess protein kinase A catalytic subunit for one hour at
30.degree. C. using the same phosphorylation conditions as
described above except that no .sup.32P-labeled ATP was present and
that after the reaction time was complete, samples were analyzed in
a fluoromiter rather than blotted onto phosphocellulose paper.
[0150] The greatest change in fluorescence occurred in mutant
214KRDSM (SEQ ID NO:15) which exhibited at 40% change in
fluorescence upon phophorylation. However. analysis of the kinetics
of phosphorylation using gamma-32P-labeled ATP demonstrated that
the site is poorly phosphorylated by protein kinase A. Wild type
GFP contains a mediocre consensus phosphorylation motif (25HKFSV,
SEQ ID NO:1) that can be phosphorylated by protein kinase A in
vitro with relatively slow kinetics. While phosphorylation at this
position has no detectable effect on the fluorescence of GFP, the
rate of phosphorylation at this position is used as an internal
control between experiments to determine the relative rates of
phosphorylation at sites engineered into the protein by site
directed mutagenesis.
[0151] B. Phosphorylation Sites At or About the N-Terminus of
Aequorea GFP
[0152] Phosphorylation sites at the N-terminus of GFP were
engineered into S65T GFP by PCR. The sequence changes, relative
fluorescence, relative rates of phosphorylation and the percent
change in fluorescence upon phophorylation are tabulated in Table
V.
8TABLE V Relative fluorescence, rate of phosphorylation and change
in fluorescence upon phosphorylation for phosphorylation sites
inserted at the N-terminus Relative fluorescence as a Relative
rates of % Change in SEQ ID NO: Sequence % of wild type
phosphorylation fluorescence SEQ ID NO.2 1MSKGEELF 100 1.0 0 SEQ ID
NO.25 1MRKGSCLF 40 5.1 5.7 SEQ ID NO.26 1MRKGSLLF 52 1.6 8.0 SEQ ID
NO.27 1MRRESLLF 30 3.0 6.0 SEQ ID NO.28 1MRDSCLF 27 3.7 17 SEQ ID
NO.29 1MSRRDSCF 43 2.1 25 SEQ ID NO.30 1MSKRRDSL 7 5.5 5.1
[0153] Numbers prior to the sequence indicate amino acid position
in the wild type GFP where the phosphorylation motif starts. The
relative rates of phosphorylation compare the rate of
phosphorylation of the given phosphorylation motif with the
endogenous protein kinase A phosphorylation motif in Aequorea GFP
(HKFSV) measured by incorporation of .sup.32P after incubation of
the purified substrate and protein kinase A catalytic subunit in
the presence of .sup.32P-labeled ATP using the standard protocols
described above. The percentage change in fluorescence represents
the change in fluorescence (488 nm excitation, 511 nm emission)
observed in each purified protein as a result of incubation with
excess protein kinase A catalytic subunit for one hour at
30.degree. C. using phosphorylation conditions described above.
These results demonstrate that mutants whose sequence closely
resembles the native protein retain considerable fluorescence,
display good kinetics of phosphorylation, but show relatively small
changes in fluorescence after phosphorylation. To improve the
effect of phosphorylation on fluorescence, amino acids around the
phosphorylation site were mutated to create an optimal
phosphorylation sequence even if it disordered the existing local
tertiary structure. Such disruption was predicted and found to
decrease the basal fluorescence of these constructs in their
non-phosphorylated state (Table VI).
9TABLE VI Relative fluorescence before phosphorylation and change
in fluorescence upon phosphorylation for more drastically altered
phosphorylation sites inserted at the N-terminus. Relative % Change
in fluor- fluor- escence escence as a % of GFP upon mutant
phosphory- SEQ ID NO: Sequence S65T lation SEQ ID NO.2 1MSKGEELF
100 0 (WILD TYPE) SEQ ID NO.31 1MSRRRSI 5.8 40 SEQ ID NO.32
1MRRRRSII 5.1 70 SEQ ID NO.33 -1MRRRRSIII N.D. 43 SEQ ID NO.34
-2MRRRRSIIIF 0.7 15 SEQ ID NO.35 -3MRRRRSIIIIF 0.6 70
[0154] Numbers prior to the sequence indicate amino acid position
in wild type GFP where the phosphorylation site starts. Negative
numbers indicate extensions onto the wild-type N-terminus. The
percent change in fluorescence represents the change in
fluorescence (488 nm excitation, 51 1 nm emission) observed in each
purified protein resulting from incubation with excess protein
kinase A catalytic subunit for one hour at 30.degree. C. using
standard phosphorylation conditions described earlier.
[0155] Perhaps because of the reduced basal fluorescence,
phosphorylation by protein kinase A produced greater percentage
increases in fluorescence in these constructs than in the more
conservative mutations of Table IV. Constructs 1MRRRRSII (SEQ ID
NO:32), MRRRRSIII (SEQ ID NO:33) and -3MRRRRSIIIIF (SEQ ID NO:35)
displayed the greatest increases, about 70%, in fluorescence upon
phosphorylation using the standard phosphorylation conditions.
However, these increased percentage increases were obtained at the
cost of reduced ability to fold at higher temperatures and
relatively poor fluorescence even after phosphorylation. To improve
these characteristics, these mutants were further optimized by
additional random mutagenesis with a novel selection procedure.
[0156] C. Further Optimization of N-Terminal Phosphorylation Sites
by Random Mutagenesis of the Remainder of GFP
[0157] The two best constructs from above (1MRRRRSII (SEQ ID NO:32)
and -3MRRRRSIIIIF (SEQ ID NO:35)) were further mutagenized and
screened for variants that were highly fluorescent when
phosphorylated, but weakly fluorescent when non-phosphorylated. The
method involved expression of a randomly mutated fluorescent
compound with or without simultaneous co-expression of the
constitutively active catalytic subunit of protein kinase A in
bacteria, and screening the individual mutants to determine those
fluorescent compounds that are highly fluorescent in the presence
but not the absence of the kinase.
[0158] To enable co-expression of the kinase and fluorescent
compound such as GFP, a new expression vector with the kinase A
catalytic subunit upstream from the fluorescent substrate was
constructed (FIG. 4). This construct enabled expression of both the
kinase and GFP from the same promoter through the insertion of a
ribosome-binding site between the coding regions of the first and
second genes. Random mutations were introduced into GFP by
error-prone PCR and the resulting population of mutants cloned into
the co-expression vector using the appropriate restriction sites.
The expression library of vectors contained the mutated fluorescent
compounds were transformed into host bacteria and individual
bacterial colonies (each derived from a single cell, and hence
containing a single unique mutant fluorescent compound) were
cultured.
[0159] The colonies were screened for fluorescence either by
fluorescence-activated cell sorting or by observation of individual
colonies grown on an agar plate under a microscope. Those colonies
that exhibited the greatest fluorescence were re-screened under
conditions in which the kinase gene was inactivated. This was
achieved in either of two ways. In the first method the
co-expression vector was isolated and treated with restriction
endonucleases and modifying enzymes (EcoR1, klenow fragment, and T4
DNA ligase) to cut the kinase gene, add additional bases and
religate the DNA, causing a frame shift and hence inactivating the
gene. The treated and non-treated plasmids were then re-transformed
into bacteria and compared in fluorescence. Alternatively, the
plasmids were initially grown in a RecA- (recombinase A negative)
bacterial strain. where the kinase is stable, to screen for
brighter mutants in the presence of the kinase. The plasmid DNA was
then isolated and re-transformed into a strain of bacteria which is
RecA+. in which the kinase is unstable and is lost through
homologous recombination of the tandomly repeated ribosome biding
sites (rbs). The bacteria have a strong tendency to eliminate the
kinase A catalytic subunit because it slows their multiplication,
so cells that splice out the kinase by recombination have a large
growth advantage.
[0160] Comparison of the brightness of the mutant first in the
presence of kinase then in its absence indicates the relative
effect of phosphorylation on the mutant GFP fluorescence (after
normalizing for GFP expression levels). A library of approximately
2.times.10.sup.6 members was screened by this approach.
Approximately 500 mutants displayed higher levels of fluorescence
when screened in the presence of the kinase. After inactivation of
the kinase, one mutant out of the 500 displayed reduced levels of
fluorescence. The increased fluorescence of the remainder of the
500 mutants was independent of the presence of the kinase. This
mutant GFP was isolated and sequenced and found to contain the
following mutations compared to wild-type GFP (SEQ ID NO:2) (in
addition to the N-terminal phosphorylation site 1MRRRRSII (SEQ ID
NO:32)): S65A, N149K, V163A and 1167T).
[0161] To confirm that this mutant was indeed directly sensitive to
protein kinase A phosphorylation and to quantify its responsively,
it was expressed in the absence of kinase. The E. coli were lysed
and the protein purified as described earlier using a nickel
affinity column. The protein exhibited high levels of fluorescence
when induced at 30.degree. C. but displayed reduced fluorescence
when incubated at 37.degree. C. After such preincubation
(37.degree. C. overnight) and separation of the less fluorescent
material by centrifugation, this protein exhibited the largest
change in fluorescence upon phosphorylation yet observed. The
tolerance of this mutant for 37.degree. C. treatment suggested that
this mutant is suitable for use in mammalian cells.
[0162] D. GFP Mutants Exhibiting Phosphorylation Dependent
Quenching
[0163] A phosphorylation recognition motif and substrate site for
protein kinase A was engineered into the N-terminal region of GFP
having the mutations S65A. N149K. V163A, and 1167T (Examples A to
C). Further mutations were made within the coding sequence of GFP
at positions that were identified to be in close three-dimensional
contact with the site of phosphorylation (phosphorylation site is
at Glu5 in the wild type protein (SEQ ID NO:2)). These mutants were
designed to strengthen ionic interactions between the phosphoserine
and internal positively charged amino acids such as Lvs79, for
example by mutation of Lys79 to Arg or His. Additional mutations
were also made to disorder the local N-terminal structure of the
GFP in the non-phosphorylated form, for example, by disrupting the
interactions between Lys3 and Glu90, by mutation of Glu90 to Lys or
Asn. These mutations were made to both enhance the effect of
phosphorylation on the fluorescent properties of GFP and to improve
the accessibility of the phosphorylation motif or site to the
kinase.
[0164] Mutation of amino acids close in sequence to the site of
phosphorylation can also be changed to further weaken their
interactions with other amino acid residues, although the sequence
around the site of phosphorylation may directly impact the
efficiency of phosphorylation by altering or disrupting the
recognition motif for phosphorylation. An example of such a change
is the mutation of Phe8 to the smaller and less hydrophobic amino
acid Leu, which can disrupt or reduce hydrophobic interactions
between Phe8 and LysS5, Cys70, Leu 19 and Met88. Also, mutation of
Gly4 to Ala provides a relatively small hydrophobic amino acid that
is preferred as a phosphorylation motif, and would not distort the
interaction between the point of phosphorylation and its point of
interaction within the GFP molecule. Not wishing to be bound to any
mechanism of action, the inventors postulate that the
phosphorylation of GFP may result in a transition from a locally
disordered to ordered state without initially causing gross changes
in protein conformation. This change can cause different
fluorescent properties of the GFP in the phosphorylated and
non-phosphorylated states under quenching conditions.
[0165] GFP mutant K4 (SEQ ID NO:49) (-2M,
-1G,M1R,S2R,K3R,G4A,E5S,E6I,L7I,- S65A,N149K,V163A,I167T) which
contains a protein kinase A substrate recognition motif and
substrate site for an activity, was used as the basis for other
mutants K5 to K16. Single mutants K79R (K5), E90N (K6), E90K (K7)
and double mutants K79R/E90N (K8), K79R/E90K (K9), K79H, E90N
(K10), K79H, E90K (K11), K79H (K12), K79E, E90N (K13), K79E, E90K
(K14), K79E (K15) and K79Q (K16) of K4 were made using known
methods (see, Sambrook, Molecular Cloning. A Laboratory Manual,
Cold Spring Harbor Press (1989)).
[0166] 1. Use of Low pH as a Quenching Agent to Enhance
Fluorescence Changes of GFP Mutants Upon Phosphorylation
[0167] Mutants K4, K5, K6, K7, K8 and K9 were evaluated for
fluorescence properties in their phosphorylated and
non-phosphorylated states as a function of pH. Individual GFP
mutants (4 micromolar) were phosphorylated by incubation with mouse
recombinant protein kinase A catalytic subunit (Calbiochem
#539-487, specific activity of 7,100 unit per milligram of protein)
(1 unit in 20 mM MOPS, pH 7.3, 1 mM DTT, 3 mM MgCl.sub.2, 1 mM ATP
at 30.degree. C. for 1 hour in a total volume of 50 microliters).
Control samples were incubated under the same conditions without
protein kinase A. All fluorescence measurements were made using the
Perseptive Biosystems 96 well plate reader with standard excitation
and emission filters (Ex 485/25. Em 530/30) and gain setting of 70.
Measurements were made approximately five minutes after addition of
100 microliters of the quenching buffer (50 mM citrate, 100 mM
NaCl) provided at the indicated pH. The results represent the means
of triplicate determinations. The fluorescence of the
phosphorylated GFP mutant relative to the non-phosphorylated GFP
mutant was calculated and presented in Table VII. These data
demonstrate that mutants can exhibit changes in a fluorescent
property upon quenching at low pH. and that their sensitivity to
quenching is different for different mutants.
10TABLE VII Effect of pH on Quenching of GFP Mutants Fold Change in
Fluorescence of GFP in the Phosphorylated State at the Indicated pH
Compared to Non-Phophorylated Samples Mutant 5.6 5.4 5.2 5.0 4.8 K4
0.84 0.86 0.91 1.04 1.92 K5 1.0 1.0 1.05 1.6 1.9 K6 1.0 1.0 1.0 1.6
2.5 K7 0.95 0.95 1.0 1.4 2.0 K8 0.90 0.90 1.0 1.4 2.8 K9 1.0 1.0
1.0 1.3 2.3
[0168] The composition of the buffer used to stabilize the pH at
the indicated value (for example, acetate, or citrate/phosphate)
had little effect on quenching. Acetate buffers provided slightly
greater and more robust changes than citrate. The highest degree of
quenching in this example occurred using 100 mM acetate buffer in
the presence of 100 mM NaCl at a volume that was twice that of the
sample (standard quenching conditions). Preferred quenching
conditions were dependent on the sample pH and the GFP mutant.
[0169] 2. Effect of Time of Incubation and Low pH on Fluorescence
of Phosphorylated and Non-Phosphorylated GFP mutant K8
[0170] The time dependency of changes in quenching were
investigated using GFP mutant K8 over a range of pH values using
standard quenching buffer (Table VIII). The procedures described
for the data presented in Table VII were used for these
experiments, except that measurements were made at the indicated
times. The "Time of Incubation" column represents the amount of
time that the samples were under quenching conditions before
fluorescence measurements were taken.
11TABLE VIII Effects of Time on Quenching of GFP Mutant K8 Fold
Increase in Time of Incubation Fluorescence of GFP in (hours)
Optimal pH the Phosphorylated Sate 0 4.6 1.5 0.5 4.8 3.3 1.5 5.0
3.7 4.5 5.0 5.1 7.5 5.0 6.1 10.5 5.0 7.5
[0171] Lower pH values of the quenching agent (for example, pH 4.6
or below) resulted in relatively smaller changes in fluorescence
compared to control samples that were maximal relatively rapidly
after addition of the quenching agent. Higher pH 1 values (4.8 to
5.0) of the quenching agent resulted in larger differences in
fluorescence, although these changes required larger times of
incubation (up to ten hours). Maximal effects of quenching with low
pH buffers were obtained around pH 5.0+0.2 with 100 mM acetate
buffer with 100 mM NaCl. Maximal effects of low pH quenching were
obtainable after ten to twenty-four hours, depending on the pH of
the quenching agent used. After this time, fluorescence differences
were stable for up to 72 hours. If the pH of the quenching agent
(100 mM sodium acetate with 100 mM NaCl) was above pH 5.4,
phosphorylation mediated fluorescence changes remained small, even
up to 24 hours of incubation These results are summarized in FIG.
5.
[0172] Quenching with low pH buffer caused a decrease in the
relative fluorescence of the non-phosphorylated GFP mutant K8
compared to the phosphorylated GFP mutant K8 (see FIG. 6).
[0173] 3. Effect of Ionic Strength, Detergents, and Organic
Solvents on Fluorescence of Phosphorylated and Non-Phosphorylated
GFP Mutants
[0174] The relative quenching of GFP mutant K8 in a phosphorylated
and non-phosphorylated state was enhanced by the presence of 100 mM
NaCl. Higher or lower concentrations of salt reduced the magnitude
and kinetics of quenching for both phosphorylated and
non-phosphorylated samples, but did enhance the relative difference
in quenching between phosphorylated and non-phosphorylated samples.
The inclusion of a divalent cation chelator such as EDTA or CDTA
stabilized the fluorescence of phosphorylated GFP mutant K8,
possibly by inhibiting acid phosphatases present as a contaminant
in the sample or buffer. Beta-glycerol phosphate (Sigma) (25 mM)
was also an effective inhibitor of acid phosphatase activities.
[0175] The detergents Triton.RTM. X-100, Tween.RTM. 20. NP-40 and
CHAPS.RTM. (in the concentration range of 0-01 to 2 percent) in 100
mM acetate buffer, 100 mM NaCl, pH 4.6 to 9.0 reduced the
fluorescence of both the phosphorylated and non-phosphorylated
samples and increased the rate of loss of fluorescence of both the
phosphorylated and non-phosphorylated samples. Based on these
results, these quenching agents can be included in the quenching
conditions to accelerate the rate of quenching which can make these
assays more convenient.
[0176] Urea and guanidine HCl (up to a concentration of 3 M) at pH
7.0 did not significantly enhance the relative quenching of
phosphorylated or non-phosphorylated GFP mutant K8.
[0177] 4. Analysis of Phosphorylation Kinetics by Radiolabel-Based
Measurements of Protein Phosphorylation
[0178] The phosphorylation kinetics of GFP mutant K8 (-2M, -1G,
M1R, S2R, K3R, G4A, E5S, E61, L7I, S65A, K79R, E90N. N149K, V163A,
1167T) and a control which lacked the N-terminal phosphorylation
motif and site present in the mutant K8 were determined using the
incorporation of .sup.32P-phosphate (FIG. 7). Experiments were
conducted using following reaction conditions: 20 mM MOPS, pH 7.4,
100 mM KCl, 0.2% Tween.RTM. 20, 2.5 units of protein kinase A.
Phosphorylation reactions were initiated by the addition of
radiolabeled ATP and magnesium (5 .mu.Ci .sup.32P-ATP per tube) to
GFP mutant K8 at a concentration of 2 micromolar. Phosphorylation
reactions were performed for the indicated times at 30.degree. C.
and were terminated by the addition of 10% trichloroacetic acid
(TCA). Bovine serum albumin was added as a carrier (10 microliters
of 1% BSA per tube) and the resulting precipitate was collected by
centrifugation. The resulting pellet was washed three times in 10%
TCA prior to counting radioactivity by Cerenkov counting. The GFP
mutant K8 exhibited greater incorporation of .sup.32P than the
control that lacks the N-terminal phosphorylation site. The results
of these experiments demonstrate that the GFP mutant K8 is rapidly
phosphorylated by protein kinase A.
[0179] Kinetic analysis of the rate of phosphorylation of GFP
mutant K8 having the phosphorylation motif set forth in SEQ ID
NO:49 measured by .sup.32P incorporation revealed an apparent Km of
9 .mu.M and a turnover number (Kcat) of 1.9 sec.sup.-1 at
30.degree. C. Analysis of these parameters based on quenching alone
gave an estimated Km of 7.7 .mu.M and Kcat of 1.2 sec.sup.-1. Thus,
both of these methods gave similar results, validating the use of
quenching alone to determine these parameters and phosphorylation
in general.
[0180] 5. Validation of GFP Mutant K8 for Use in 96 Well
Homogeneous Fluorescence-Based Kinase Assays.
[0181] The limit of detectability of protein kinase A using the
GFP-based fluorescent assay set forth in these examples in a 96
well assay format was determined by measuring the fluorescence
changes in response to incubation with a range of protein kinase A
concentrations, as is described in Table VII. Reactions were
performed under standard conditions for forty minutes at 30.degree.
C. Fluorescent measurements were made using a Cytofluor 2 series
4000 from Perseptive Biosystem 96 well plate reader fitted with
standard excitation and emission filters (485/25, 530/30) and set
at a gain of 70. Assays were performed using Costar 96 well black
plates with clear bottom in a reaction volume of 50 microliters.
Reactions were terminated by addition of the preferred acetate
quenching conditions (100 mM acetate buffer pH 5.0, 100 mM NaCl, 25
mM Beta-glycerol phosphate ).
[0182] At the lowest concentration of PKA tested (26 pmol, or 0.5
ng) a detectable change in fluorescence signal was observed upon
quenching. Fifty-two pmol of PKA gave an approximately two-fold
increase in fluorescence compared to controls that were incubated
in the absence of PKA. These results demonstrate that the assay
provides highly sensitive measurements.
[0183] 6. Detecting GFP Mutant K8 Using 96-well plate reader.
[0184] These instrument settings and plates were used to determine
the limit of detection of GFP mutant K8. In TRIS buffer at pH 8.0,
the GFP mutant K8 was detectable above background fluorescence at
approximately 0.1 .mu.M. After treatment with the preferred
quenching conditions at pH 5.0 GFP mutant K8 was detectable above
background fluorescence at a limiting concentration of about about
0.5 .mu.M. These results demonstrate that the GFP mutant K8 can be
detected with high sensitivity using standard 96-well plate readers
in a typical screening environment.
[0185] 7. Robustness of the Assay to the Effects of
Co-Solvents.
[0186] Assay robustness to co-solvents is a highly desirable
feature of drug screening systems because many drug candidates are
not appreciably soluble in aqueous solutions. The co-solvents DMSO
or ethanol are frequently used in drug screening at concentrations
up to about 1% to dissolve drugs or target compounds. Thus, it is
important to establish that these agents alone do not significantly
influence the quenching of GFP at these concentrations. The
preferred assay conditions as described above were used to
determine the effects of the solvents DMSO and ethanol on the GFP
mutant K8 phosphorylation assay.
[0187] DMSO or ethanol were added at 0, 0.01, 0.1, 0.2, 0.5, and
10% (Vol/Vol) to the kinase reaction mixture. At the maximum
concentrations tested, these agents exhibited little or no effect
of phosphorylation on the fluorescence development after quenching.
These results establish that co-solvents such as DMSO or ethanol at
concentrations used in screening assays do not interfere
appreciably with GFP mutant K8 quenching assays.
[0188] 8. Assay Validation with Protein Kinase A Inhibitors.
[0189] The protein kinase A inhibitors PKI (protein kinase A
heat-stable inhibitor, isoform alpha (Calbiochem #539488) and H-89
(N-[2-((p-bromocinrinamyl)amino)ethyl]-5-isoquinoline sulfonamide,
HCl) (Calbiochem #371963) were tested in the preferred assay
condition to determine if they could be detected using the GFP
mutant K8 quenching assay. Both compounds were tested individually
(between 0.1 and 1,000 nM) and pre-incubated with PKA in the
absence of mutant K8 for 10 minutes at 4.degree. C. Then, GFP
mutant K8 was added to a final concentration of 4 .mu.M and the
mixtures were incubated for thirty minutes at 30.degree. C. The
kinase reactions were terminated by the addition of the preferred
acetate quenching conditions (100 mM acetate, 100 mM NaCl. pH 5.0,
25 mM beta-glycerol phosphate). The fluorescence of these samples
was measured 14 to 16 hours later. The results of these studies are
presented in FIG. 8. The results of these studies establish that
the GFP mutant K8 quenching assay can be used to detect kinase
inhibitors and further validates the methodology for drug
screening.
[0190] 9. Detection of ATP Antagonists Using GFP Mutant K8
Quenching Assay
[0191] The protein kinase A inhibitor H-89 was tested in the GFP
mutant K8 kinase assay at two different ATP concentrations (0.1 mM
and 1 mM). In the presence of the higher ATP concentration, the
inhibitor was much less efficient at inhibiting kinase activity.
The response of the inhibitor to different ATP concentrations
indicates that it acts by inhibiting ATP binding to the kinase
active site. Because the GFP kinase assay can be performed at both
high and low ATP concentrations, this method can be used to
identify, measure, and detect ATP antagonists. Furthermore assay
conditions can be established (i.e. the use of high or low ATP
levels) to select for, or screen out, such compounds. This provides
a significant improvement over competing assay technologies such as
radioactive incorporation that can only be run at high sensitivity
with low ATP concentrations. The results of these studies establish
that the GFP mutant K8 quenching assay can be used to detect kinase
inhibitors and further validates the methodology.
[0192] 10. Use of GFP to Measure Other Kinase Activities.
[0193] In addition to GFP mutant K8 having a PKA phosphorylation
recognition motif, the inventors have made versions of GFP mutant
K8 that have phosphorylation recognition motifs that are selective
for protein kinase C (PKC) and mitogen activated protein kinase
(MAP) (also known as extracellular regulated kinase (erk) (Table
IX)). These substrates have the same site of phosphorylation as GFP
mutant K8, which corresponds to Glu5 in the wild-type protein.
12TABLE IX Additional protein phosphorylation motifs introduced
into GEP mutant K8. Relative Fluorescence Compared to Clone GFP
mutuant Kinase Specifity Phosphorylation Motif Name K8
Corresponding wild-type Met Ser Lys Gly Glu Glu Len Phe (SEQ ID
NO.36) Wild type sequence Erk Kinase Met Val Glu Pro Leu Thr Pro
Ser Phe (SEQ ID NO.64) Erk-1 1.40 Erk Kinase Met Thr Gly Pro Leu
Ser Pro Gly Phe (SEQ ID NO.65) Erk-4 1.49 Erk Kinase Met Thr Gly
Pro Leu Ser Pro Gly Tyr (SEQ ID NO.66) Erk-5 1.26 Erk Kinase Met
Thr Gly Pro Leu Ser Pro Gly Leu (SEQ ID NO.67) Erk-6 1.22 Erk
Kinase Met Thr Gly Pro Leu Ser Pro Gly Pro (SEQ ID NO.68) Erk-7
0.40 PKC .alpha. Arg Arg Arg Arg Arg Lys Gly Ser Phe Arg (SEQ ID
NO.56) Pkc 3 0.72 PKC .alpha. (+Membrane Arg Arg Arg Arg Arg Lys
Gly Ser Phe Arg (SEQ ID NO.57) Pkc 3 lys 0.95 association motif
(hepta- Lys) at the C-terminus)) PKC .beta.1 Phe Lys Leu Lys Arg
Lys Gly Ser Phe Lys (SEQ ID NO.58) Pkc 4 1.1.3 PKC .delta. Ala Arg
Arg Lys Arg Lys Gly Ser Phe Phe (SEQ ID NO.59) Pkc 5 1.91 PKC
.epsilon. Tyr Tyr Ala Lys Arg Lys Met Ser Phe Phe (SEQ ID NO.60)
Pck 6 1.09 PKC .zeta. Arg Arg Phe Lys Arg Gln Gly Ser Phe Phe (SEQ
ID NO.61) Pkc 7 1.88 PKC .mu. Ala Ala Leu Val Arg Gln Met Ser Val
Ala (SEQ ID NO.62) Pkc 8 1.84
[0194] MAPKs (for mitogen-activated protein kinases) or ERKs (for
extracellular-regulated kinases) selective phosphorylation motifs
were introduced into GFP mutant K8 based on their known preferred
substrate recognition motifs. Phosphorylation motifs were designed
based on Songyang et al., Mo. Cell Biol. 16:6486-6493 (1996) and
were made by replacing the protein kinase A phosphorylation motif
in the GFP mutant K8 with the indicated phosphorylation motif using
PCR methods known in the art. PKC isoform specific phosphorylation
motifs were based on the sequences identified by Nishikawa et al.
J. Biol. Chem. 272:952-960 (1997). These phosphorylation motifs
were introduced into GFP mutant K8 by PCR as described above.
[0195] All of the constructs were successfully expressed at high
level and were highly fluorescent (Table IX). The ERK substrates
showed no substantial sequence identity with either the wild-type
GFP or the PKA motifs present in GFP mutant K8, yet in most cases
were as fluorescent or more fluorescent than GFP mutant K8. These
results demonstrate that many different N-terminal phosphorylation
motifs can be successfully introduced into GFP without
significantly impacting GFP fluorescence.
[0196] E. Rates and Efficiencies of Phosphorylation of Additional
GFP Substrates
[0197] 1. Erk Selective Substrates
[0198] Table X reports the phosphorylation rates of various GFP
mutant sensors containing Erk selective recognition motifs. The
constructs Erk-6 and Erk-7 exhibited the greatest rates of
phosphorylation. Kinetic analysis of the Erk-7 construct revealed a
Km of 15 .mu.M and a Kcat of 0.055 sec.sup.-1.
13TABLE X Phosphorylation rates of various GFP Kinase Sensors
Sample .sup.32P-Incorporation (CPM) Control 102 .+-. 32 Myelin
Basic Protein 33361 .+-. 477 Erk-1 1579 .+-. 282 Erk-4 1982 .+-.
260 Erk-5 6455 .+-. 558 Erk-6 22498 .+-. 381 Erk-6-B17 13,499 .+-.
472 Erk-7 25502 .+-. 2077
[0199] These studies were performed by incubating GFP or MBP
(myelin basic protein) (10 .mu.M) with activated recombinant MAP
kinase (Calbiochem #454855) (100 ng) for 30 minutes at 30.degree.
C. in buffer (20 mM MOPs, pH 7.2, 25 mM .beta.-glycerol phosphate,
5 mM EGTA, 1 mM DTT, 0.1 mM ATP, 20 mM MgCl.sub.2 10 .mu.Ci
.sup.32P-ATP in a volume of 50 microliters). Experiments were
terminated and samples processed as described previously in
subsection D4. Results presented in Table X represent means of
triplicate determinations.+-.standard deviations of
.sup.32P-incorporation in washed pellets as described earlier.
[0200] The value of Kcat obtained for the GFP Erk substrate was
similar to the Kcat values obtained for myelin basic protein, a
well-characterized substrate of Erk kinase. These results
demonstrate that Erk-1 selective phosphorylation motifs can be
introduced into GFP and that the site of phosphorylation is rapidly
and efficiently phosphorylated, with comparable kinetics to other
proteins or peptides that are known substrates of Erk-1
kinases.
[0201] a. Mutagenesis of Erk Selective Substrates Erk-6 and Erk-7
to Improve Fluorescence and Kinetics of Phosphorylation.
[0202] To improve the fluorescent properties of the Erk kinase
substrates (for example, Erk-6 and Erk-7), a library of mutants
derived from these clones was made in which amino acids in the
interior of the GFP that interact (with the three-dimensional
structure of the protein) with the N-terminal region of GFP were
mutated. The mutants were designed to produce a "better fit" of the
Erk phosphorylation motif into the top of the barrel of GFP. This
was achieved by enhancing the size of the positive charge
associated around the site of phosphorylation (by mutation of K85
to R), pushing the backbone amide chain closer to the N-terminal
phosphorylation recognition motif (by mutation of A87 to larger
amino acids) and by making E90 more hydrophobic so that it could
attract Pro 3 and therefore move closer to the phosphorylation
motif. This library of mutants were screened for improved
brightness and folding. Mutagenesis resulted in the creation of
better folding, and a more fluorescent version, of the Erk-6
mutant, but did not significantly improve the fluorescence or
folding of the Erk-7 mutant.
[0203] Selected clones from the Erk-6 and Erk-7 mutagenesis
reactions were sequenced to confirmed that mutagenesis was
successfully accomplished. The non-wild type sequences are
displayed in TABLE XI. Therein, poorly fluorescent clones exhibited
less than 10% of the fluorescence of GFP mutant K8 and were not
further characterized. The GFP mutant Erk-6-B 17 showed 158% of the
fluorescence of GFP mutant K8, demonstrating that the mutagenesis
approach was successful in improving GFP fluorescence.
14TABLE XI Mutants of Erk-6 and Erk-7 Substrates. Mutant Name
Mutations Fluorescence E6-B17 A87T, E90A Highly fluorescent E6-A5
K85R Poorly Fluorescent E6-A10 K85R, A87T, E90L Poorly Fluorescent
E6-A14 K85R, A87V, E90P Poorly Fluorescent E6-A17 K85R, A87T, E90S
Poorly Fluorescent E7-B19 E901 Poorly Fluorescent E7-B22 A87T, E90R
Poorly Fluorescent E7-A40 K85R, A87T, E90N Poorly Fluorescent
E7-A42 K85R, A87T, E90P Poorly Fluorescent
[0204] b. Effect of Phosphorylation on the Fluorescence Changes in
GFP After Quench.
[0205] Analysis of the effect of quenching on the fluorescence of
the mutants Erk-6, Erk-7 and Erk-6-B 17 was performed. The results
presented in Table XII demonstrate improved fluorescent changes in
response to quenching with acetate buffer. These results
demonstrate that the mutagenesis approaches are generally
applicable to improve fluorescence and phosphorylation dependent
changes in quenching.
[0206] Table XII: Effects of quenching on the fluorescence of Erk-6
and Erk-7 mutants
15TABLE XII Effects of quenching on the fluorescence of Erk-6 and
Erk-7 mutants Fold Increase in Fluorescence Constructs after
Phosphorylation Erk-6 1.20 Erk-6-17B 1.40 Erk-7 1.05
[0207] These experiments were performed by incubating the GFP
sample (2 .mu.M) with Erk-1 kinase (1 .mu.g) for 1 hour at
30.degree. C. in assay buffer (20 mM MOPS. pH 7.2, 25 mM
beta-glycoerol phosphate, 5 mM EGTA. 1 mM DTT, 1 mM ATP, 20 mM
MgCl.sub.2). Reactions were quenched by the addition of acetate
quenching buffer (100 .mu.L of 100 mM Acetate pH 5.0, 100 mM NaCl,
20 mM beta-Glycerol phosphate). Fluorescence changes were measured
after 3 hours of incubation in acetate quenching buffer. Results
represent the means of triplicate determinations.
[0208] The mutant Erk-6-B17 exhibited 1.4 times greater
fluorescence than the original construct Erk-6. Incubation of this
mutant with an excess activated kinase resulted in larger change in
fluorescence after quenching. These results demonstrate that these
methods of improving mutants are generally applicable to the
creation and improvement of a range of phosphorylation motifs.
[0209] F. PKC Selective Phosphorylation Motifs
[0210] The rates of phosphorylation of GFP having PKC motifs were
determined by measuring .sup.32P incorporation in the presence of
different PKC isoforms (Table XIII). These examples include one
example where a membrane association motif is part of the GFP
mutant.
16TABLE XIII Rate of Phosphorylation of Various GFP Kinase
Substrates Using Various Kinases Kinase and Activity (CPM) GFP
Mutant K8 Having the Indicated Phosphorylation Motif PKC alpha PKC
.epsilon. PKC .zeta. PKC alpha 10,629 21,734 8,129 PKC alpha +
Membrane 22,675 10,230 2,138 Association motif (Hepta-Lys) PKC
.beta.1 13,332 39,533 20,173 PKC .delta. 4,935 12,473 7,733 PKC
.epsilon. 11,310 43,705 26,783 PKC .zeta. 4,745 12,688 8,421 PKC
.mu. 5,230 20,259 14,606
[0211] These experiments were performed by incubating the indicated
GFP mutant (5 micromolar) with the indicated PKC isoform (0.2
.mu.g) for 30 minutes at 30.degree. C. in 25 mM TRIS pH 7.5. 1 mM
DTT, 10 mM MgCl.sub.2 0.1 mM ATP 20 .mu.g/ml phosphatidylserine, 10
.mu.M OAG, 200 .mu.M CaCl.sub.2 (for PKC.alpha.) and 1 mM EGTA (for
PKCs .epsilon. and .zeta.). These results demonstrate that the
available sequence diversity available at the N-terminus of GFP is
sufficient to generate isoform specific phosphorylation of
different mutants. The relative specificities identified for the
GFP substrates in this experiment broadly matched those identified
by Nishikawa et al (1997) who used non-GFP peptides to selectively
measure PCK isoform activity. GFP contains an endogenous
phosphorylation site (underlined) (Gly His Lys Phe Ser Val Ser Gly)
within a relatively poorly recognized phosphorylation recognition
motif that may be phosphorylated by some PKC isoforms. This may
reduce the apparent specificity of the N-terminal phosphorylation
motifs as measured by .sup.32P-incorporation because these data
represent phosphorylation both at the N-terminal site and the
internal site. Membrane association motif poly-Lys (hepta-Lys) was
added to the C-terminus of GFP mutant K8 with PKC alpha
phosphorylation motif at the N-terminus.
[0212] 1. Determination of Fluorescence Changes in Response to
Phosphorylation
[0213] To determine if changes in fluorescence correlated with
changes in phoshorylation, the previous experiments were repeated
except that the fluorescence changes rather than .sup.32P
incorporation were measured after the addition of a quenching
agent. These results demonstrate that fluorescence changes in the
GFP samples after quenching correlate with the incorporation of
.sup.32P (see Table XIV).
[0214] Maximal quenching for the PKC substrates was observed with
50 mM acetate buffer at pH 5.2 in the presence of 20% DMSO. The
maximum change in fluorescence observed was typically 1.6 to 2.0
fold greater fluorescence for the phosphorylated substrate after 24
hours under the best conditions identified. The difference in
quench conditions for the case of the PKC specific substrates
compared to the PKA substrate may be due to the large hydrophobic
motif C-terminal to the site of phosphorylation in these substrates
(Ser-Phe-(Phe/Arg)-Phe).
[0215] 2. Addition of Membrane Association and Protein-Protein
Interaction Motifs to GFP
[0216] A polybasic membrane association motif derived from K-ras
(Hancock et al. EMBO J. 12:4033-4039 (1991))) (Hepta-Lys) was added
to the C-terminus of the PKC-alpha GFP by PCR. In addition, the
farnesyl modification site could be added to the Hepta-Lys motif,
resulting in the sequence
Lys-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys-Thr-Lys-Cys-Val-Ile-Met (SEQ ID
NO:63) to create a tighter membrane association. The
phosphorylation kinetics of this GFP was compared to that of a GFP
that contained the PKC-alpha specific phosphorylation motif, but
not the membrane associated motif. Both the constructs were highly
fluorescent and were expressed at high levels in bacteria. The
putative membrane associated GFP was soluble in aqueous solution at
high salt concentration (0.3 M NaCl), but precipitated upon storage
after dialysis to 0.1 M NaCl. All experiments using this protein
were conducted on material that was stored in high salt and diluted
into low ionic strength media in the presence of phospholipid
vesicles immediately prior to experiments.
[0217] The addition of a membrane association motif significantly
increased the rate of phosphorylation of the substrate compared to
a GFP substrate with the same phosphorylation recognition motif,
but lacking the membrane association motif (FIG. 9). The addition
of the membrane recognition motif also had a significant effect on
the specificity of the PKC alpha with respect to other PKC isoforms
(Table XIII). Kinetic analysis of the PKC alpha substrates with or
without the membrane association motif reveals that increased
phosphorylation was primarily due to an increase in apparent Km of
the substrate, with little effect on the Vmax (FIG. 9).
[0218] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
Sequence CWU 0
0
* * * * *
References