Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation Number:7,176,037 from the United States Patent and Trademark Office (PTO) owispatent The invention provides peptide synthons having protected functional groups for attachment of desired moieties (e.g. functional molecules or probes). Also provided are peptide conjugates prepared from such synthons, and synthon and conjugate preparation methods including procedures for identifying optimum probe attachment sites. Biosensors are provided having functional molecules that can locate and bind to specific biomolecules within living cells. Biosensors can detect chemical and physiological changes in those biomolecules as living cells are moving, metabolizing and reacting to its environment. Methods are included for detecting GTP activation of a Rho GTPase protein using polypeptide biosensors. When the biosensor binds GTP-activated Rho GTPase protein, an environmentally sensitive dye emits a signal of a different lifetime, intensity or wavelength than when not bound. New fluorophores whose fluorescence responds to environmental changes are also provided that have improved detection and attachment properties, and that can be used in living cells, or in vitro.<H intermediates, preparation, Labeled, peptide, 7176037.html, Patent, pto, 7176037

Title: Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation

Abstract: The invention provides peptide synthons having protected functional groups for attachment of desired moieties (e.g. functional molecules or probes). Also provided are peptide conjugates prepared from such synthons, and synthon and conjugate preparation methods including procedures for identifying optimum probe attachment sites. Biosensors are provided having functional molecules that can locate and bind to specific biomolecules within living cells. Biosensors can detect chemical and physiological changes in those biomolecules as living cells are moving, metabolizing and reacting to its environment. Methods are included for detecting GTP activation of a Rho GTPase protein using polypeptide biosensors. When the biosensor binds GTP-activated Rho GTPase protein, an environmentally sensitive dye emits a signal of a different lifetime, intensity or wavelength than when not bound. New fluorophores whose fluorescence responds to environmental changes are also provided that have improved detection and attachment properties, and that can be used in living cells, or in vitro.

Patent Number: 7,176,037 Issued on 02/13/2007 to Hahn, et al.

Inventors: Hahn; Klaus M. (San Diego, CA), Toutchkine; Alexei (San Diego, CA), Muthyala; Rajeev (Champaign, IL), Kraynov; Vadim (San Diego, CA), Bark; Steven J. (San Diego, CA), Burton; Dennis R. (La Jolla, CA), Chamberlain; Chester (Cambridge, MA)
Assignee: The Scripps Research Institute (La Jolla, CA)

Appl. No.: 10/455,713

Filed: June 3, 2003

Current U.S. Class: 436/546 ; 435/7.1; 436/172; 436/544; 530/403; 548/152

Current International Class: G01N 33/533 (20060101); C07D 277/62 (20060101); C07K 1/13 (20060101); G01N 21/76 (20060101); G01N 33/53 (20060101); G01N 33/532 (20060101)

Field of Search: 436/544,546,172 530/333,387.3,388.9,402,391.3,403 435/7.1 548/152

References Cited [Referenced By]

U.S. Patent Documents


3578456	May 1971	Petro et al.
3764322	October 1973	Kampfer et al.
3810760	May 1974	Kampfer et al.
3862111	January 1975	Low et al.
4469768	September 1984	Hori et al.
4750228	June 1988	Naef
4816567	March 1989	Cabilly et al.
4994356	February 1991	Diehl et al.
5268486	December 1993	Waggoner et al.
5880270	March 1999	Berninger et al.
6001364	December 1999	Rose et al.
6180295	January 2001	Helber et al.
6331385	December 2001	Deaton et al.
6951947	October 2005	Hahn et al.
2002/0055133	May 2002	Hahn, et al.
2005/0287518	December 2005	Hahn et al.

Foreign Patent Documents


969284	Jan., 2000	EP
0985970	Mar., 2000	EP
WO-0075105	Dec., 2000	WO

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Primary Examiner: Lee; Long V.
Assistant Examiner: Haq; Shafiqul
Attorney, Agent or Firm: Schwegman, Lundberg, Woessner, & Kluth, P.A.

Government Interests

GOVERNMENT FUNDING

The invention described herein was made with United States Government support under Grant Number MCB-9812248 awarded by the National Science Foundation, and under Grant Numbers CA58689, AG15430 and GM 57464 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US01/22194, filed on Jul. 13, 2001, which claimed priority under 35 U.S.C. 119 of U.S. application Ser. No. 09/839,577, filed Apr. 20, 2001, U.S. Provisional Application Ser. No. 60/279,302, filed Mar. 28, 2001, PCT/US00/26821, filed Sep. 29, 2000, and U.S. Provisional Application Ser. No. 60/218,113, filed Jul. 13, 2000, which applications are incorporated herein by reference.

Claims

What is claimed:

1. A method of detecting the location or environment of a cellular protein within a living cell which comprises: a. introducing a biosensor into the living cell wherein the biosensor binds to a tag on the cellular protein; and b. detecting the location or environment of a functional molecule on the biosensor within the living cell; wherein said tag is a peptide segment that has been fused to the cellular protein and said biosensor comprises the peptide conjugate of formula (III) or (IV): ##STR00014## wherein R.sup.6 is a peptide, a polypeptide or an antibody; X is a direct bond or a linking group; R.sup.7 is hydrogen, (C.sub.1 C.sub.6)alkyl, an amino protecting group, or a radical comprising one or more aminooxy groups; Y is a direct bond or a linking group; Z is a linking group, or Z is a direct single bond or a double bond between N and D; and D is a functional molecule of the formula: ##STR00015## wherein: each m is separately an integer ranging from 1 3; n is an integer ranging from 0 to 5; R.sup.8, R.sup.11 and R.sup.12 are separately CO, SO.sub.2, C.dbd.C(CN).sub.2, S, O or C(CH.sub.3).sub.2; each R.sup.13 is alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability; each R.sup.9 and R.sup.10 is separately an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules; provided that when D is a peptide, N and D are not linked through a --C.dbd.N--O--CH.sub.2--C(.dbd.O)-- linkage in the backbone of the peptide conjugate.

2. A method of detecting the location or environment of a cellular protein within a living cell which comprises: a. introducing a biosensor into the living cell wherein the biosensor binds to a tag on the cellular protein; and b. detecting the location or environment of a functional molecule on the biosensor within the living cell; wherein said tag is a peptide segment that has been fused to the cellular protein, and said functional molecule is a fluorescent compound of the formula: ##STR00016## wherein: each m is separately an integer ranging from 1 3; n is an integer ranging from 0 to 5; R.sup.8, R.sup.11 and R.sup.12 are separately CO, SO.sub.2, C.dbd.C(CN).sub.2, S, O or C(CH.sub.3).sub.2; each R.sup.13 is alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability; each R.sup.9 and R.sup.10 is separately an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.

3. The method of claim 1 or 2 wherein said tag has SEQ ID NO:16.

4. The method of claim 1 or 2 wherein said cellular protein is calmodulin, Rho GTPase, rae, cdc42, mitogen-activated protein kinase, Erk1, Erk2, Erk3, Erk4, IgE receptor (F.sub.cRI) actin, .alpha.-actinin, myosin, or a major histocompatibility protein.

5. The method of claim 2, wherein said functional molecule is a compound having any one of the following formulae: ##STR00017## ##STR00018##

6. A method of detecting the location or environment of a cellular protein within a living cell which comprises: (a) introducing a biosensor into the living cell wherein the biosensor binds to a tag on the cellular protein; and (b) detecting the location or environment of a functional molecule on the biosensor within the living cell; wherein said tag is a peptide segment comprising SEQ ID NO:16 (KMAQLKKKVQALKSKVASLKKKVQALKKKVAQR-NH2); and wherein the functional molecule is a fluorescent dye of the formula: ##STR00019## wherein: each m is separately an integer ranging from 1 3: n is an integer ranging from 0 to 5: R.sup.8, R.sup.11 and R.sup.12 are separately CO, SO.sub.2, C.dbd.C(CN).sub.2, S, O or C(CH.sub.3).sub.2; each R.sup.13 is alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability; each R.sup.9 and R.sup.10 is separately an alkyl chain derivatized with charged groups to enhance water solubility or with reactive groups for conjugation to other molecules.

Description

BACKGROUND OF THE INVENTION

Modified peptides and proteins are valuable biophysical tools for studying biological processes, both in vitro and in vivo. They are also useful in assays to identify new drugs and therapeutic agents. In particular, quantitative live cell imaging using fluorescent proteins and peptides is revolutionizing the study of cell biology. An exciting recent development within this field has been the construction of peptide and protein biosensors exhibiting altered fluorescence properties in response to changes in their environment, oligomeric state, conformation upon ligand binding, structure, or direct ligand binding. Appropriately labeled fluorescent biomolecules allow spatial and temporal detection of biochemical reactions inside living cells. See for example Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405 434; Day, R. N., Mol. Endocrinol. 1998, 12:1410 9; Adams, S. R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature 1997, 388:882 7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K. M., et al., J. Biol. Chem. 1990, 265:20335; and Richieri, G. V., et al., Mol. Cell. Biochem. 1999, 192:87 94.

Procedures for site-specific modification of polypeptides have been described, including: chemically selective labeling in solution (Brinkley, M. Bioconjugate Chemistry 1992, 3:2 13) and on resin bound peptides (Hackeng, T., et al., J. Biol. Chem. Submitted); introduction of ketone amino acids through synthetic procedures (Rose, K. J. Am. Chem. Soc. 1994, 116:30 33; King, T. P., et al., Biochemistry, 1986, 25:5774 5779; Rose, K., et al., Bioconjugate Chem. 1996, 7:552 556; Marcaurelle, L. A., Bertozzi, C. R. Tett. Lett. 1998, 39:7279 7282; and Wahl, F., Mutter, M. Tett. Lett. 1996, 37:6861 6864); and molecular biology techniques (Cornish, V. W., et al., J. Am. Chem. Soc. 1996, 118:8150). For example, green fluorescence protein (GFP) is a fluorescent protein that has been fused to other proteins using molecular biology techniques and has been used to visualize intracellular proteins (see, e.g., Katz et al., BioTechniques 25: 298 304 (1998)).

While each of these methods has utility for producing a particular class of biosensor or labeled polypeptide, all have limitations that restrict their general use. Labeling of natural amino acid side-chains in solution is often impractical because of the existence of many other competing nucleophiles. Additionally, the use of unnatural amino acids, such as those bearing ketones for selective labeling, requires the synthesis of dye constructs or amino acids that are difficult to make and are not available commercially. While a variety of proteins have been fused to GFP, some GFP-labeled proteins fail to fluoresce. Mutational analysis indicates that the structure of GFP is extremely sensitive to molecular or biochemical modifications. Moreover, GFP is extremely large (238 amino acids). Hence, fusion of GFP to other proteins can alter the function of GFP or even the function of the protein to which it is fused. Moreover, GFP does not have the diverse capabilities of smaller, synthetic molecules, some of which provide a variety of fluorescence wavelengths, or are capable of reporting on protein conformation, or can photo-crosslink or act as NME or EPR probes. Accordingly, new fluorescent molecules with more diverse properties and new attachment methods are needed.

Currently, the major obstacles to the development of fluorescent biosensors and labeled polypeptides remain: (1) The difficulty in site-specific placement of the dye in the polypeptide and (2) determining exactly which site is optimal for dye placement (Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405 434). Solvent-sensitive dyes and other biophysical probes must be placed precisely for optimal response to changes in protein structure without interference with biological activity. Also, the need for site-specific incorporation of two dyes without impairment of biological activity has proven a serious limitation for utilization of fluorescence resonance energy transfer (FRET) within a single protein. Total chemical synthesis of proteins provides a potential solution to these problems (Wilken, J., Kent, S. B. H. Curr. Op. Biotechnology. 1998, 9:412; Kent, S. B. H. Ann. Rev. Biochem. 1988, 57, 957 989; Dawson, P. E., et al., Science 1994, 266:776 779; Muir, T. W., et al., Proc. Natl. Acad. Sci. 1998, 95:6705 6710; and Cotton, G. J., et al., J. Am. Chem. Soc. 1999, 121:1100 1101). However, many biophysical probes suitable for fluorescent biosensors or other purposes are not stable to the various conditions used for peptide synthesis, and site-specific incorporation after synthesis has been difficult to achieve.

Moreover, labeling with hydrophobic dyes such as thionine or methylene blue can be problematic because these dyes autoaggregate in aqueous solution at high concentration. See, J. Am. Chem. Soc. 63, 69 (1941). These aggregates cause a change in the absorption spectrum and a reduction in the fluorescence of the dyes. Cyanines and merocyanines are also thought to aggregate, causing a quenching of fluorescence (J. Phys. Chem. 69, 1894 (1965)). Such aggregation interferes with conjugation of these fluorescent dyes to other molecules such as proteins. Moreover aggregation by cyanines and merocyanines can be exacerbated after the dyes are conjugated. For example, Waggoner et al. have observed an aggregation phenomenon following the conjugation of cyanin isothiocyanate with an antibody (Cytometry 10, 11 19 (1989)). Fluorescence of a conjugate between a cyanin fluorescent dye and an anti-HCG antibody (molar ratio=1.7) named CY5.18 is quenched in comparison with that of the free cyanin (see U.S. Pat. No. 5,268,486 and Anal. Biochem. 217, 197 204 (1994)). Also, while cyanines are generally stable, inexpensive, simple to conjugate to other molecules and of a suitable size for the recognition of small molecules, they do not change their fluorescence in response to environmental factors, such as solvent polarity. New dyes that eliminate these problems are needed.

Thus, there is currently a need for new fluorescent dyes and peptide synthons having protected functional groups that can be selectively modified to incorporate one or more functional molecules (e.g. a fluorescent label) following peptide synthesis. There is also a need for proteins and antibodies with biophysical probes attached to precise locations, and for simple, non-destructive methods of making such labeled proteins and antibodies. Simpler methods for using these labeled peptides, proteins and antibodies in vivo as biosensors are also needed.

SUMMARY OF THE INVENTION

The present invention provides a highly efficient method for the site-specific attachment of biophysical probes or other molecules to unprotected peptides following chemical synthesis. The methodology utilizes amino acids having one or more protected aminooxy groups, which can be incorporated during solid-phase peptide synthesis or which can be combined with recombinant peptides through post expression steps. It has been discovered that the protected aminooxy group can be unmasked following peptide synthesis, and reacted with an electrophilic reagent to provide a modified (e.g. a labeled) peptide. The aminooxy group reacts selectively with electrophiles (e.g. an activated carboxylic ester such as an N-hydroxy-succinimide ester) in the presence of other nucleophilic groups including cysteine, lysine and amino groups.

Thus, selective peptide modification (e.g. labeling) can be accomplished after synthesis using commercially available and/or chemically sensitive molecules (e.g. probes). The methodology is compatible with the synthesis of C.sup..alpha.-thioester containing peptides and amide-forming ligations, required steps for the synthesis of proteins by either total chemical synthesis or expressed protein ligation. An aminooxy containing amino acid can be introduced into different sites by parallel peptide synthesis to generate a polypeptide analogue family with each member possessing a single specifically-labeled site. The parallel synthesis enables the development of optimized biosensors or other modified polypeptides through combinatorial screening of different attachment sites for maximal response and minimal perturbation of desired biological activity.

Thus, a simple and efficient methodology for site-specific modification (e.g. labeling) of peptides after synthesis has been developed that provides high yield, selectivity, and compatibility with both solid-phase peptide synthesis and C.sup..alpha.-thioester peptide recombinant synthesis.

Accordingly, the invention provides a synthetic intermediate (i.e. a synthon) useful for preparing modified peptides, which is a compound of formula (I):

##STR00001## wherein: R.sup.1 is hydrogen or an amino protecting group; R.sup.2 is hydrogen or a carboxy protecting group; R is an organic radical comprising one or more aminooxy groups.

Peptides including one or more aminooxy groups are also useful synthetic intermediates that can be modified to provide related peptides having altered biological, chemical, or physical properties, such as, for example, a peptide linked to a fluorescent label. Accordingly, the invention also provides a peptide having one or more (e.g. 1, 2, 3, or 4) aminooxy groups; provided the peptide is not glutathione. The invention also provides a peptide having one or more (e.g. 1, 2, 3, or 4) secondary aminooxy groups.

The invention generally provides intermediates and methods that allow for site-specific modification of peptides after synthesis. Accordingly, functional molecules can be selectively linked to a peptide to provide a peptide conjugate having altered biological, chemical, or physical properties. For example, functional molecules (e.g. biophysical probes, peptides, polynucleotides, and therapeutic agents) can be linked to a peptide to provide a peptide conjugate having differing and useful properties.

Thus, the invention also provides a compound of formula (III):

##STR00002## wherein:

R.sup.6 is a peptide, polypeptide or antibody;

X is a direct bond or a linking group;

R.sup.7 is hydrogen, (C.sub.1 C.sub.6)alkyl, an amino protecting group, or a radical comprising one or more aminooxy groups;

Y is a direct bond or a linking group; and

D is a functional molecule.

A functional molecule can be any label, dye, pharmaceutical, toxin, Preferably the functional molecule is a biophysical probe, such as a fluorescent group that can be used for FRET studies or other studies involving fluorescent signals, such as excimer pair formation.

Processes for preparing synthons of the invention as well as the polypeptide, antibody and protein conjugates of the invention are provided as further embodiments of the invention and are illustrated by the procedures in the Examples below.

Thus, the invention also provides a method for preparing a peptide conjugate comprising a peptide and a functional molecule, comprising reacting a peptide having one or more aminooxy groups with a corresponding functional molecule having an electrophilic moiety, to provide the peptide conjugate.

The present invention further provides environment-sensing dyes that can be readily conjugated to proteins and other molecules without the problems of aggregation, fluorescence quenching and the like. The present dyes strongly fluoresce and can fluoresce in an environmentally-sensitive manner suitable for use in living cells. Unlike cyanine dyes, the environmental sensitivity of these dyes can be used to form biosensors that can report many aspects of protein behavior, or the behavior of other molecules. Protein behaviors including conformational change, phosphorylation state, ligand interaction, protein-protein binding and various post-translational modifications affect the distribution of charged and hydrophobic residues can be reported by the present dyes by changes in their fluorescence.

The present invention overcomes the disadvantages of the available environmentally-sensitive fluorescent dyes. The present fluorescent probes exhibit high fluorescence levels before and after conjugation to other molecules, including peptides, proteins and antibodies, and changes in fluorescence suitable for many purposes, including in vivo and in vitro assays of protein behavior.

The present invention provides new fluorescent dyes that can be used in any manner chosen by one of skill in the art. The dyes can be linked to any useful molecule known to one of skill in the art using any available procedure. In one embodiment the fluorescent dyes are linked to peptides, polypeptides or antibodies using the methods provided herein. These dyes have the following structure (IV).

##STR00003## wherein:

each m is separately an integer ranging from 1 3;

n is an integer ranging from 0 to 5;

R.sup.8, R.sup.11 and R.sup.12 are separately CO, SO.sub.2, C.dbd.C(CN).sub.2, S, O or C(CH.sub.3).sub.2;

each R.sup.13 is alkyl, branched alkyl or heterocyclic ring derivatized with charged groups to enhance water solubility and enhance photostability;

R.sup.9 and R.sup.10 are chains carrying charged groups to enhance water solubility (i.e. sulfonate, amide, ether) and/or chains bearing reactive groups for conjugation to other molecules. The reactive group is a functional group that is chemically reactive (or that can be made chemically reactive) with functional groups typically found in biological materials, or functional groups that can be readily converted to chemically reactive derivatives using methods well known in the art. In one embodiment of the invention, the charged and reactive groups are separately haloacetamide (--NH--(C.dbd.O)--CH.sub.2--X), where X is Cl, Br or I. Alternatively, the charged and reactive groups are separately amine, maleimide, isocyanato (--N.dbd.C.dbd.O), isothiocyanato(--N.dbd.C.dbd.S), acyl halide, succinimidyl ester, or sulfosuccinimidyl ester. In another embodiment, the charged and reactive groups are carboxylic acid (COOH), or derivatives of a carboxylic acid. An appropriate derivative of a carboxylic acid includes an alkali or alkaline earth metal salt of carboxylic acid. Alternatively, the charged and reactive groups are reactive derivatives of a carboxylic acid (--COORx), where the reactive group Rx is one that activates the carbonyl group of --COORx toward nucleophilic displacement. In particular, Rx is any group that activates the carbonyl towards nucleophilic displacement without being incorporated into the final displacement product. Examples of COORx: ester of phenol or naphtol that is further substituted by at least one strong electron withdrawing group, or carboxylic acid activated by carbodiimide, or acyl chloride, or succinimidyl or sulfosuccinimidyl ester. Additional charged and reactive groups include, among others, sulfonyl halides, sulfonyl azides, alcohols, thiols, semicarbazides, hydrazines or hydroxylamines.

The invention still further provides a method of identifying an optimal position for placement of a functional molecule on a peptide having a peptide backbone and a known activity, which includes making a series of peptide conjugates, each peptide conjugate having the same amino acid sequence and the same functional molecule, wherein the functional molecule is linked at a different location along the backbone of every peptide conjugate in the series, and observing which functional molecule location does not substantially interfere with the known activity of the peptide.

The invention also provides a method of identifying an optimal position for placement of a functional molecule in a protein having a known activity and an identified peptide segment for attachment of the functional molecule, which includes making a series of peptide conjugates, each peptide conjugate having the amino acid sequence of the identified peptide segment and the same functional molecule, wherein the functional molecule is linked at a different location along the backbone of every peptide conjugate in the series; replacing the identified peptide segment in each protein of a series of said proteins with a peptide conjugate selected from the series of peptide conjugates to create a series of protein conjugates each having the functional molecule at a different location; and observing which functional molecule location does not substantially interfere with the known activity of the protein.

The invention further provides a method of identifying an optimal position for placement of an environmentally-sensitive functional molecule on a peptide biosensor having a backbone, which includes making a series of peptide conjugates, each peptide conjugate having the same amino acid sequence and the same functional molecule, wherein the functional molecule is at a different location along the backbone of every peptide conjugate in the series, and observing which functional molecule location provides the strongest signal change in response to an environmental change in the peptide conjugate. The signal change can be any observable change in a signal, for example, the change can be a change in fluorescence emission intensity, fluorescence duration or fluorescence emission wavelength. The environmental change in the peptide biosensor can be, for example, an interaction with a target molecule.

The invention still further provides a method of identifying an optimal position for placement of an environmentally-sensitive functional molecule in a protein having a known activity and an identified peptide segment for attachment of the functional molecule, which includes making a series of peptide conjugates, each peptide conjugate having the amino acid sequence of the identified peptide segment and the same environmentally-sensitive functional molecule, wherein the environmentally-sensitive functional molecule is linked at a different location along the backbone of every peptide conjugate in the series; replacing the identified peptide segment in each protein of a series of said proteins with a peptide conjugate selected from the series of peptide conjugates to create a series of protein conjugates, each having the environmentally-sensitive functional molecule at a different location; and observing which functional molecule location provides the strongest signal change in response to an environmental change in the protein conjugate.

The invention also provides a method for detecting GTP activation of a Rho GTPase protein, which includes contacting a polypeptide biosensor with a test substance, wherein said polypeptide biosensor comprises a polypeptide capable of binding a GTP-activated Rho GTPase protein, and wherein said polypeptide is operatively linked to an environmentally sensitive fluorescent dye; and observing fluorescence emissions from the polypeptide biosensor at a wavelength emitted by said fluorescent acceptor dye; wherein the environmentally sensitive fluorescent dye will emit light of a different intensity or a different wavelength when the polypeptide biosensor is bound to the GTP-activated Rho GTPase protein than when the polypeptide biosensor is not bound.

The invention further provides a method of detecting the location of a cellular protein within a living cell that includes providing the living cell with a biosensor capable of binding to a tag on the cellular target protein; and detecting the location of a functional molecule on the biosensor within the living cell. The tag on the cellular target protein can be a peptide segment that has been fused to the cellular protein. In one embodiment, the tag is a peptide which includes SEQ ID NO:16 and that can bind to a biosensor having a peptide segment with SEQ ID NO:15. The biosensor can include a peptide-conjugate of the invention. Any of the functional molecules, pharmaceuticals, toxins, labels, dyes or compounds can also be present on the biosensor. Moreover, any cellular protein can be detected using this method, for example, calmodulin, Rho GTPase, rac, cdc42, mitogen-activated protein kinase, Erk1, Erk2, Erk3, Erk4, IgE receptor (F.sub.c.epsilon.RI, actin, .alpha.-actinin, myosin, or a major histocompatibility protein. The methods provided herein can detect and identify the cellular location of proteins and cellular proteins that have never been successful labeled and observed in vivo.

BRIEF DESCRIPTION OF THE FIGURES

In the following detailed description of example embodiments of the invention, reference is made to the accompanying drawings which form a part hereof, and which is shown by way of illustration only, specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

FIG. 1 illustrates a general strategy for site-specific labeling of polypeptides. The protected aminooxy group is incorporated during solid-phase peptide synthesis (synthesis on a thioester-linker resin is shown); cleavage from the resin generates a peptide possessing unprotected sidechains, an aminooxy group and a C-terminal thioester; and ligation and subsequent site-specific labeling produces the full-length peptide with a functional molecule attached at the aminooxy nitrogen.

FIG. 2 illustrates the synthesis of PA-test and SA-test peptides.

FIG. 3 shows HPLC analysis of purified SA-test peptide (top) and crude reaction products from optimized labeling conditions (bottom).

FIG. 4 illustrates the synthesis of a protected intermediate (4) of the invention.

FIG. 5 illustrates one method for using biosensors according to the present invention. In this example, the activation of Rac1 ("RAC") by GTP was observed. A fragment of p21-Activated kinase (PAK) capable of binding to Rac1 (termed "PBD") was used as a biosensor because PAK will only bind to Rac1 when Rac1 is activated by GTP. A Rac1-Green Fluorescent Protein fusion protein (shown as a square named "RAC" attached to a circle named "GFP") was made and a cell line expressing this fusion protein was generated. Cells expressing RAC-GFP were injected with PBD labeled with Alexa-546 dye ("Dye"). The PBD biosensor binds selectively to GTP-RAC-GFP, but not to GDP-RAC-GFP. Upon binding to GTP-RAC-GFP, the Alexa on the labeled fragment undergoes fluorescence resonance energy transfer (FRET) as the Alexa and GFP fluorophores are brought close together. This FRET can be measured within a living cell or in vitro to map the distribution, localization and level of Rac-GTP activation. FRET produces a fluorescence signal which is distinct from a GFP fluorescence signal because energy is transferred from the excited GFP fluorophore to the nearby Alexa dye (J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Plenum Press, New York, 1983), pp. 305 341)). By imaging the cell with different wavelengths, both the distribution of Rac and Rac activation can be studied in the same cell. GFP excitation and emission are used for overall Rac distribution, while GFP excitation and Alexa emission are used for FRET.

FIG. 6 illustrates one method of using an environmentally sensitive fluorescent dye in the present methods so that changes in naturally existing proteins can be detected and observed in vivo or in vitro.

FIG. 6a depicts FRET between the Rac1-Green Fluorescent Protein (GFP-Rac1) fusion and the p21-activated kinase biosensor (PBD) labeled with Alexa-546 dye as shown in FIG. 5. Inactive Rac1 is depicted as a larger gray circle with Green Fluorescent Protein (smaller circle) attached. Upon activation by GTP, Rac1 undergoes a structural change depicted as a gray circle changing to a half-rounded gray rectangle. Unbound PBD is depicted as a black L-shape with an attached Alexa-546 dye (open circle). Before Rac1 is activated, PBD cannot bind and the Alexa-546 cannot undergo FRET. However, after Rac1 activation, the Rac1 assumes a conformation that permits PBD binding. Such binding juxtaposes the Green Fluorescence Protein and the Alexa-546, which produces FRET.

FIG. 6b illustrates how an environmentally sensitive fluorescent dye eliminates the need to create a fusion protein like the GFP-Rac1 protein depicted in FIG. 6a. In FIG. 6b, a natural, unmodified protein is depicted as a gray oval. The protein changes conformation upon activation by GTP, depicted as the transition to a half-rounded gray rectangle. When the protein is in the activated state, a polypeptide-biosensor that binds only to the activated state (black L-shape), with an attached environmentally sensitive dye (open circle), can bind. Upon binding, the environmentally sensitive dye will emit light of a different wavelength, duration or intensity (filled circle) than before binding. Use of this type of environmentally sensitive dye is further illustrated in the Figures to follow.

FIG. 7 illustrates what conditions will optimally provide FRET between GFP-Rac and Alexa-PBD in vitro. FIG. 7A shows fluorescence emission from solutions containing a fixed level of GFP-Rac bound to GTP.gamma.S at different concentrations of Alexa-PBD (PBD labeled with Alexa-546). Light at 480 nm was selectively used for GFP excitation, and direct (non-FRET) excitation of Alexa was subtracted from these spectra. In the absence of Alexa-PBD, the emission from GFP (peak at 508 nm) is maximal and no Alexa emission (peak at 568 nm) is seen. As the concentration of Alexa-PBD is increased, binding of Alexa-PBD to Rac-GFP leads to FRET, producing increasing emission at 568 nm and a decrease at 508 nm. FIG. 7B shows the variation of the 568/508 nm emission ratios with changes in the level of GTP (solid circles) or GDP (solid squares). All data points were the average of three independent experiments.

FIG. 8 illustrates how GFP-Rac expression levels and levels of intracellular Alexa-PBD (as observed by fluorescence) correlate with changes in normal cell behavior produced by these proteins. FIG. 8A shows what levels of GFP-Rac expression, as measured by log GFP intensity per cell area, were correlated with ruffling. Cells with different expression levels of either wild type or a constitutively active Q6 IL mutant of GFP-Rac were scored for ruffling. Each point represents an individual cell, placed in the higher (Ruffling) or lower (Nonruffling) row depending on whether ruffling was induced. As illustrated, there is a level of GFP intensity below which ruffling was consistently not induced by expression of wild type GFP-Rac. Only cells with Rac expression levels below 250 on this scale were used in biological experiments. The validity of this approach was supported by scoring of constitutively active Rac, which showed ruffle induction at much lower levels of expression.

FIG. 8B shows which levels of intracellular Alexa-PBD would perturb normal serum-induced ruffling, as observed by Alexa intensity per cell area. Cells were scored as in FIG. 8A, at different levels of Alexa-PBD fluorescence. Based on this experiment, only cells with Alexa-PBD concentrations below 400 intensity units on this scale were used in biological experiments. These-studies demonstrated that FRET could still be readily detected at appropriately low levels of introduced protein.

FIG. 9 illustrates the dynamics of Rac activation during growth factor stimulation of quiescent cells. FIG. 9A provides photomicrographs showing Rac localization (GFP-Rac) and Rac activation (FRET) before stimulation of quiescent Swiss 3T3 fibroblasts. FIG. 9B provides photomicrographs of the same Swiss 3T3 fibroblasts three minutes after addition of serum. Warmer or lighter colors correspond to higher intensity values. The cells showed accumulation of Rac at and around the nucleus before stimulation (GFP-Rac image). Most of the nuclear GFP-Rac was associated with the nuclear envelope. Serum or PDGF addition generated multiple moving ruffles that showed FRET, while no FRET was seen at the nucleus before or after stimulation. Of thirty-five cells stimulated with either serum or PDGF, thirty-one began ruffling within 15 minutes. FRET was seen in the ruffles of all but one of the ruffling cells.

FIGS. 9C and 9D demonstrate that simple localization of Alexa-PBD is inferior to FRET in quantifying and localizing Rac-GTP binding (Bar=8 .mu.m). The ruffle in FIG. 9B is shown in close-up in FIG. 9C, visualized using FRET. PBD localization in the same region is visualized using Alexa fluorescence in FIG. 9D, with scaling optimized for detection of the ruffle. Without prior knowledge of the ruffle's location, this localization would have been difficult to discem. The high background due to unbound PBD cannot be eliminated in FIG. 9D and binding to other target proteins is not eliminated as it is in the highly specific FRET signal.

In each of the GFP-Rac images above, intensities range between 300 1100. The image of FRET before serum addition was scaled to demonstrate the low levels of FRET, with values ranging between 0 and 15. In the image of FRET after stimulation, the ruffle contains the highest values of 40 to 65. Nuclear FRET was not seen in any of the cells examined.

FIG. 10 illustrates Rac activation in motile Swiss 3T3 fibroblast cells. This figure shows two examples of where a Rac 1 activation gradient is formed, in confluent monolayer cells and in "wound healing" cells. High Rac1 activation occurred at the leading edge of motility, particularly in wound healing cells. In these experiments high levels of Rac-GTP were frequently seen in the juxtanuclear region of the cell (FIG. 10A). The strong correlation of this gradient with the direction of movement indicates that activated Rac is spatially organized in polarized cells to help guide or propagate movement. Comparison of the GFP (FIG. 10A) and FRET (FIG. 10B) images shows that the distribution of activated Rac does not parallel that of Rac 1 itself. FRET intensities (FIG. 10B) are 0 18 (top image) and 0 32 (bottom image). In the GFP images (FIG. 10A), intensities range between 98 700 (top image) and 100 1100 (bottom image).

FIG. 11 provides the structure of one of the present fluorescent dyes and the spectrum of fluorescence emission for that dye in water, methanol and butanol. As illustrated, the fluorescent emission of this dye increases with increasing solvent hydrophobicity. This figure illustrates the environmental-sensitivity of this dye.

FIG. 12 provides the structure of another fluorescent dye of the present invention and shows its spectrum in aqueous solution, compared to similar dyes lacking the groups designed to prevent aggregation. In the curve from each dye, the peak to the right is the unconjugated, highly fluorescent form of the dye, while that to the left is the weakly fluorescent form. The curve furthest to the right is the dye containing groups to prevent aggregation. As illustrated, the chosen groups help prevent aggregation of the dye.

FIG. 13 provides one method for synthesizing a dye of the present invention. Conversion of compound 1 to an amine 2 followed by protection of the amine provides compound 3, which can be alkylated to give compound 4. Reaction of compound 4 with compound 9 provides compound 5, which can be deprotected to provide amine 6. Alkylation or acylation of amine 6 with a chain carrying a charged group, or with a chain bearing a reactive group for conjugation to another molecule, or with another molecule directly provides a dye of the invention 7. Intermediate compound 9 can be prepared by condensation of compound 8 with the requisite aldehyde, under conditions that are known in the art

FIG. 14 provides a three-dimensional image of the CRIB domain ("CBD") of the Wiscott-Aldrich syndrome protein (WASP) bound to Cdc42. The essential residues are depicted in yellow, hydrophobic residues depicted in purple and the red sites show positions where the new dyes were attached to generate changes in fluorescence when the CBD bound Cdc42.

FIG. 15 provides the intensity of fluorescence at various wavelengths observed when fluorescently labeled CBD binds to cdc42. When cdc42 is activated with GTP.gamma.S, highly intense fluorescence is observed (line labeled "GTP.gamma.S"), compared to when no cdc42 is present (line labeled "no cdc42"), or when cdc42 is not activated (line labeled "GDP").

FIG. 16 shows how the present methods can be used for in vitro assays on crude cellular lysates. In this case, the fluorescently labeled CBD was added to a neutrophil lysate. Upon binding to activated cdc42, CBD will emit fluorescence of greater intensity. At time zero fMLP, which stimulates neutrophils to activate cdc42, was added to the cellular lysate and the amount of fluorescence generated