Oxidation reduction sensitive green fluorescent protein variants Number:7,015,310 from the United States Patent and Trademark Office (PTO) owispatent

Title: Oxidation reduction sensitive green fluorescent protein variants

Abstract: The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineered to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

Patent Number: 7,015,310 Issued on 03/21/2006 to Remington,   et al.

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Inventors:	Remington; S. James (Eugene, OR); Hanson; George T. (Madison, WI)
Assignee:	The State of Oregon Acting by and through the State Board of Higher Education (Eugene, OR)
Appl. No.:	471857
Filed:	March 11, 2002
PCT Filed:	March 11, 2002
PCT NO:	PCT/US02/07374
371 Date:	March 8, 2004
102(e) Date:	March 8, 2004
PCT PUB.NO.:	WO02/077011
PCT PUB. Date:	October 3, 2002

Current U.S. Class:	530/350; 530/300; 530/350; 435/7.1; 435/69.1; 435/325; 435/252.3; 435/320.1; 514/2; 514/12; 514/21
Current Intern'l Class:	C07K 1/00     (20060101)
Field of Search:	530/350,300 435/71,691,325,252.3,320.1 514/12,2,21

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References Cited [Referenced By]

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Other References

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Primary Examiner: Kerr; Kathleen M.
Assistant Examiner: Robinson; Hope
Attorney, Agent or Firm: Klarquist Sparkman LLP

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Goverment Interests

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STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grant number GM07759-22 and grant number GM42618-10 both awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATIONS

This is a § 371 U.S. National Stage of International Application No. PCT/US02/07374, filed Mar. 11, 2002 (published in English under PCT Article 21(2)), which in turn claims the benefit of U.S. Provisional Patent Application Nos. 60/275,200, filed Mar. 12, 2001, 60/293,427, filed May 23, 2001, and 60/302,894, filed Jul. 3, 2001.

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Claims

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We claim:

1. An isolated mutant green fluorescent protein (GFP), with a fluorescence spectrum that is sensitive to redox status,

wherein said mutant GFP shares at least 90% sequence identity with SEQ ID NO: 1 and

wherein mutations at positions corresponding to SEQ ID NO: 1 include:

a) having a cysteine at one or both of the residues corresponding to 147 or 149 of SEQ ID NO: 1 and

b) having a cysteine at one or both of the residues corresponding to 202 or 204 of SEQ ID NO: 1.

2. The mutant GFP of claim 1, wherein the mutations are selected from the group consisting of:

a) the residues corresponding to 147 and 202 of SEQ ID NO: 1 are cysteine;

b) the residues corresponding to 147 and 204 of SEQ ID NO: 1 are cysteine;

c) the residues corresponding to 149 and 202 of SEQ ID NO: 1 are cysteine;

d) the residues corresponding to 149 and 204 of SEQ ID NO: 1 are cysteine; and

e) the residues corresponding to each of residues 147, 149, 202, and 204 of SEQ ID NO: 1 are cysteine.

3. The mutant GFP of claim 1, comprising an additional mutation with respect to SEQ ID NO: 1 corresponding to position 65.

4. The mutant GFP of claim 3, wherein the residue corresponding to position 65 in SEQ ID NO: 1 is threonine.

5. The mutant GFP of claim 1, wherein the fluorescence spectrum is also pH sensitive.

6. The mutant GFP of claim 1, comprising an additional mutation with respect to SEQ ID NO: 1 corresponding to position 48.

7. The mutant GFP of claim 6, wherein the residue corresponding to position 48 in SEQ ID NO: 1 is serine.

8. The mutant GFP of claim 1, comprising additional mutations with respect to SEQ ID NO: 1 corresponding to positions 48, 65, 149, and 202,

wherein the residues corresponding to positions 48, 65, 149, and 202 are serine, threonine, cysteine, and cysteine, respectively.

9. The mutant GFP of claim 1, comprising additional mutations with respect to SEQ ID NO: 1 corresponding to positions 48, 65, 149, and 204,

wherein the residues corresponding to positions 48, 65, 149, and 204 are serine, threonine, cysteine, and cysteine, respectively.

10. The mutant GFP of claim 1, comprising additional mutations with respect to SEQ ID NO: 1 corresponding to positions 48, 147, and 204,

wherein the residues corresponding to positions 48, 147, and 204 are serine, cysteine, and cysteine, respectively.

11. The mutant GFP of claim 1, comprising additional mutations with respect to SEQ ID NO: 1 corresponding to positions 48, 147, and 202,

wherein the residues corresponding to positions 48, 147, and 202 are serine, cysteine, and cysteine, respectively.

12. The mutant GFP of claim 1, comprising additional mutations with respect to SEQ ID NO: 1 corresponding to positions 48, 65, 147, 149, 202, and 204,

wherein the residues corresponding to positions 48, 65, 147, 149, 202, and 204 are serine, threonine, cysteine, cysteine, cysteine, and cysteine, respectively.

13. The mutant GFP of claim 1, comprising additional mutations with respect to SEQ ID NO: 1 corresponding to positions 48, 147, 149, 202, and 204,

wherein the residues corresponding to positions 48, 147, 149, 202, and 204 are serine, cysteine, cysteine, cysteine, and cysteine, respectively.

14. A method of analyzing an oxidation-reduction condition of or in a cell comprising:

expressing the mutant GFP of claim 1 in the cell; and

measuring fluorescence signal from the mutant GFP, thereby analyzing an oxidation-reduction condition of or in a cell.

15. The method of claim 14, wherein the mutant GFP is expressed as a fusion protein.

16. The method of claim 14, further comprising analyzing a pH condition of or in the cell using the mutant GFP.

17. The mutant GFP of claim 1, comprising an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13.

18. A mutant GFP, with a fluorescence spectrum that is sensitive to redox status,

wherein said mutant GFP shares at least 90% sequence identity with SEQ ID NO: 1 and includes mutations selected from the group consisting of:

a) the residues corresponding to 147 and 204 of SEQ ID NO: 1 are cysteine and cysteine, respectively;

b) the residues corresponding to 65, 147 and 204 of SEQ ID NO: 1 are threonine, cysteine, and cysteine respectively;

c) the residues corresponding to 149 and 202 of SEQ ID NO: 1 are cysteine and cysteine, respectively;

d) the residues corresponding to 65, 149 and 202 of SEQ ID NO: 1 are threonine, cysteine, and cysteine respectively;

e) the residues corresponding to 147, 149, 202 and 204 of SEQ ID NO: 1 are cysteine, cysteine, cysteine and cysteine, respectively; and

f) the residues corresponding to 65, 147, 149, 202 and 204 of SEQ ID NO: 1 are threonine, cysteine, cysteine, cysteine and cysteine, respectively.

19. A mutant GFP, with a fluorescence spectrum that is sensitive to redox status,

wherein said mutant GFP shares at least 95% sequence identity with SEQ ID NO: 1 and includes mutations selected from the group consisting of:

a) the residues corresponding to 147 and 204 of SEQ ID NO: 1 are cysteine and cysteine, respectively;

b) the residues corresponding to 65, 147 and 204 of SEQ ID NO: 1 are threonine, cysteine, and cysteine respectively;

c) the residues corresponding to 149 and 202 of SEQ ID NO: 1 are cysteine and cysteine, respectively;

d) the residues corresponding to 65, 149 and 202 of SEQ ID NO: 1 are threonine, cysteine, and cysteine respectively;

e) the residues corresponding to 147, 149, 202 and 204 of SEQ ID NO: 1 are cysteine, cysteine, cysteine and cysteine, respectively; and

f) the residues corresponding to 65, 147, 149, 202 and 204 of SEQ ID NO: 1 are threonine, cysteine, cysteine, cysteine and cysteine, respectively.

20. An isolated mutant GFP, with a fluorescence spectrum that is sensitive to redox status,

wherein said mutant GFP shares at least 95% sequence identity with SEQ ID NO: 1 and

wherein mutations at positions corresponding to SEQ ID NO: 1 include:

a) having a cysteine at one or both of the residues corresponding to 147 or 149 of SEQ ID NO: 1 and

b) having a cysteine at one or both of the residues corresponding to 202 or 204 of SEQ ID NO: 1.

21. The mutant GFP of claim 20, wherein the mutations are selected from the group consisting of:

a) the residues corresponding to 147 and 202 of SEQ ID NO: 1 are cysteine;

b) the residues corresponding to 147 and 204 of SEQ ID NO: 1 are cysteine;

c) the residues corresponding to 149 and 202 of SEQ ID NO: 1 are cysteine;

d) the residues corresponding to 149 and 204 of SEQ ID NO: 1 are cysteine; and

e) the residues corresponding to each of residues 147, 149, 202, and 204 of SEQ ID NO: 1 are cysteine.

22. The mutant GFP of claim 20, comprising an additional mutation with respect to SEQ ID NO: 1 corresponding to position 65.

23. The mutant GFP of claim 22, wherein the residue corresponding to position 65 in SEQ ID NO: 1 is threonine.

24. The mutant GFP of claim 20, wherein the fluorescence spectrum is also pH sensitive.

25. The mutant GFP of claim 20, comprising an additional mutation with respect to SEQ ID NO: 1 corresponding to position 48.

26. The mutant GFP of claim 25, wherein the residue corresponding to position 48 in SEQ ID NO: 1 is serine.

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Description

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FIELD

The present disclosure relates to the field of genetic engineering, and in particular to green fluorescent protein (GFP) mutants that can be used to detect oxidation-reduction state, or a change in oxidation-reduction state.

BACKGROUND

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish, Aequorea victoria, has been used extensively in molecular and cell biology as a fluorescent marker. It is a 238 amino acid protein that generates its own fluorescent chromophore. The spontaneous generation of the chromophore is achieved by cyclization of the internal Ser65-Tyr66-Gly67 sequence followed by oxidation of Tyr 66 in the presence of molecular oxygen (Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994). The overall fold of the protein consists of an 11-stranded β-barrel capped by α-helices at both ends and contains a coaxial α-helix from which the chromophore is generated (Brejc et al., Proc. Natl. Acad. Sci. USA 94:2306-2311, 1997; Ormö et al., Science 273:1392-1395, 1996; Yang et al., Nat. Biotech. 14:1246-1251, 1996). GFP is unique among light emitting proteins, because it does not require the presence of any cofactors or substrates for the production of green light.

Wild-type GFP has absorption maxima at 398 and 475 nm (Morise et al., Biochemistry 13:2656-2662, 1974). Excitation at either of these wavelengths leads to emission of green light at 508 nm (Morise et al., 1974). The usefulness of GFP has been greatly enhanced by the availability of mutants with a broad range of absorption and emission maxima (Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994; Ormö et al., Science 273:1392-1395, 1996). These mutants have made possible multicolor reporting of cellular processes by allowing for the simultaneous observation of two or more gene products labeled with different colored GFP variants (Rizzuto et al., Curr. Biol. 6:183-188, 1996). In addition, fluorescence resonance energy transfer (FRET) experiments using different colored GFP's have been used to study protein-protein interactions in vivo (Heim et al., Curr. Biol. 6:178-182, 1996; Mitra et al., Gene 173:13-17, 1996).

More recently, GFP variants have been shown to be sensitive to pH (Wachter et al., Biochemistry 36:9759-9765, 1997; Elsliger et al., Biochemistry 38:5296-5301, 1999). As a consequence, they have been used as noninvasive intracellular pH indicators. For instance, Kneen et al. employed the GFP mutant S65T/F64L to determine the pH of the cytoplasm of CHO and LLC-PK1 cell lines (Kneen et al., Biophys. J. 74:1591-1599, 1998). Since GFP is genetically encoded, it can be specifically targeted to various subcellular compartments, which is a task not possible with small molecule fluorescent dyes (Llopis et al., Proc. Natl. Acad. Sci. USA 95:6803-6808, 1998). Therefore, Llopis and co-workers used the GFP variant S65G/S72A/T302Y/H231L, which has an increased pK_a, to measure the alkaline pH of mitochondria, golgi, and the cytosol of HeLa cells and rat neonatal cardiomyocytes (Llopis et al., 1998). These reports were the first to show that GFP variants could be used as biosensors and not just simple fluorescent markers. However, more recently GFP has been shown to be sensitive to halide ions and through a fusion with calmodulin, GFP's fluorescence can also vary in response to calcium ion concentration (Wachter et al., Curr. Biol. 9:R628-R629, 1999; Miyawaki et al., Proc. Natl. Acad Sci. USA 96:2135-2140, 1999).

Oxidation-reduction (redox) processes are very important in living organisms. The formation of disulfide bonds during protein folding relies upon a well maintained redox buffering system of glutathione and oxidized glutathione (Carothers et al., Arch. Biochem. Biophys. 268:409-425, 1989). There also exists a thioredoxin-like family of enzymes that catalyze the formation and isomerization of disulfide bonds in proteins (Debarbieux and Beckwith, Cell 99:117-119, 1999). In addition, redox signaling during apoptosis has been implicated in activating mitochondrial permeability transition, leading to cytochrome c release (Hall, Eur. J. Clin. Invest. 29:238-245, 1999). Redox changes in the form of cellular oxidation have also been suggested to be a final step in the apoptotic process leading to degradation of apoptotic bodies (Cai and Jones, J. Bioenerg. Biomemb. 31:327-334, 1999). Given the importance of in vivo processes such as protein folding and apoptosis that are dependant upon redox status, a non-invasive, convenient method for studying redox changes within living cells is needed.

Current methods of determining in vivo redox status have many limitations. Many present techniques require cells to be harvested before their contents can be analyzed. This type of procedure is not only very invasive but is also not a very accurate measure of the in vivo state of the cells. Moreover, it would be impossible with this technique to monitor redox changes within the same cell over a period of time. Recently, Keese et al., (Keese et al., FEBS Lett. 447:135-138, 1999) have developed an indicator of redox state in which glutathione reductase crystals were microinjected into the cytosol of human fibroblasts, and by detecting a color change of the crystals, they were able to determine the redox potential of the cytosol to be more reducing than -0.270 V. While this method may allow redox determination within single living cells, the cumbersome nature of the technique is still a major drawback. The most reasonable protocol for determining redox status is probably still that of Hwang et al. (Hwang et al., Science 257:1496-1502, 1992). They employed the tetrapeptide N-Acetyl-Asn-Tyr-Thr-Cys-NH₂ to measure the ratio of thiol to disulfide in the cytosol and secretory pathway of cultured cells. They concluded that the cytosol is more reducing than the secretory pathway with an approximate redox potential of -0.221 to -0236 V for the cytosol compared to -0.170 to -0.185 V for the secretory pathway. However, is method still required harvesting of the cells and like all the other methods, it is very labor intensive. Moreover, this technique determined redox potentials based only upon the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), potentially ignoring other redox buffering components.

SUMMARY OF THE DISCLOSURE

To overcome disadvantages of available methods for determining redox status in cells, GFP mutants (also referred to as redox sensitive GFP (rosGFP) variants) have been designed and are described herein, which can detect or "sense" changes in oxidation-reduction potentials. The rosGFP variants have been engineered to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other.

To overcome disadvantages of available methods for determining redox status in cells, GFP mutants (also referred to as redox sensitive GFP (rosGFP) variants) have been designed and are described herein, which can detect or "sense" changes in oxidation-reduction potentials. The rosGFP variants have been engineering to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other.

Examples of the provided GFP variants have ratiometric dual-excitation fluorescent properties as a function of redox state, with apparent redox potentials of -0.272 to -0.299 V.

Specific embodiments include rosGFP mutants that differ from wild-type GFP in that they comprise at least the following amino acid substitutions:

• (a) S147C/Q204C
• (b) S65T/S147C/Q204C
• (c) N149C/S202C
• (d) S65T/N149C/S202C
• (e) S147C/N149C/S202C/Q204C
• (f) S65T/S147C/N149C/S202C/Q204C
  The rosGFP mutants that include the S65T substitution are sensitive to pH as well as redox status. Particular provided mutation proteins include those referred to herein as rosGFP1, rosGFP2, rosGFP3, rosGFP4, rosGFP5, and rosGFP6.

Also provided are nucleic acid molecules encoding rosGFPs, including the specific listed rosGFPs. Optionally, these nucleic acid molecules can be functionally linked to expression control sequence(s) (such as a promoter), and/or integrated into a vector. Nucleic acid molecules encoding a rosGFP can be used to transform host cells (such as bacterial, plant, or animal cells); such transformed cells are also provided.

The disclosure also provides methods of using rosGFPs to analyze the redox status of a cell, or a subcellular compartment within a cell. In certain embodiments, both the redox status and pH of the cell (or subcellular compartment or other environment) are monitored concurrently.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1 is a fluorescence spectra graph, which shows how the fluorescence of rosGFP2 varies in response to changes in redox potential. The spectra show two excitation peaks, one near 400 nm and the other at about 490 nm, with a clear isosbestic point separating them.

FIG. 2 shows the titration of rosGFP2 with dithiothreitol. The apparent redox potential is -0.279 volts.

FIG. 3 is a graph showing the in vivo redox changes in fluorescence intensity of rosGFP2, in response to the addition of vitamin in K₃. After 30 total minutes, the addition of dithiothreitol elicits the opposite response as indicated by the reduced ratio.

FIG. 4 shows an SDS-PAGE analysis that reveals the intracellular disulfide linkage in rosGFP2. Lanes 1-6, control (C48S/S65T) and lanes 8 -13 rosGFP2 were incubated with 1 μM CuCl2 (with or without 2 mM N-ethylmaleimide; lanes 2, 3, 11, 12) or with 1 mM DTT (with or without 2 mM N-ethylmaleimide; lanes 5, 6, 8, 9). Lane 7 shows approximate molecular weights in kDa. Lanes 4 and 10 were empty.

FIG. 5. Absorbance and fluorescence excitation spectra of rosGFP2 at various redox states. The absorbance spectra (A) show the conversion of the neutral (band A; 400 mn) to the anionic (band B; 490 nm) chromophore species over time in the presence of 1 mM DTT. Band A is maximized under oxidizing conditions, whereas band B is favored under reducing conditions. Fluorescence spectra (B) were collected at various redox potentials and also show the interconversion of chromophore charge states. Absorbance and fluorescence spectra were both normalized to the intensity of the fully reduced protein.

FIG. 6. Fluorescence excitation spectra of rosGFP4 as a function of redox potential. The entire spectrum (A) shows the redox potential dependence on the excitation spectra of rosGFP4. Expanded the region around 400 nm (B), reveals a well resolved isosbestic point. Fluorescence intensity values were normalized to the maximum intensity at E_o′-0.320 V and emission was monitored at 510 nm.

FIG. 7. Fluorescence excitation spectra of rosGFP6 as a function of redox potential. Fluorescence intensity values were normalized to the maximum intensity at E_o′-0.310 V and emission was monitored away from the peak at 535 nm.

FIG. 8. Absorbance and fluorescence excitation spectra of oxidized rosGFP2 as a function of pH. Absorbance scans (A) were taken on samples of rosGFP2 containing 0.5 μM CuCl₂ at the indicated pHs. These samples were then diluted in the same buffer and their fluorescence excitation spectra (B) were collected. Fluorescence intensity values were normalized to the maximum intensity at pH 9.0 and emission was monitored at 510 nm.

FIG. 9. pH titration of oxidized and reduced rosGFP2. Absorbance values at 490 nm (band B) were plotted versus pH for oxidized (A) and reduced (B) rosGFP2. The data were then fitted to a titration curve with a single pK_a value.

FIG. 10. Absorbance and fluorescence excitation spectra of reduced rosGFP2 as a function of pH. Absorbance scans (A) were taken on samples of rosGFP2 containing 1 mM DTT at the indicated pHs. These samples were then diluted in the same buffer and their fluorescence excitation spectra (B) were collected. Fluorescence intensity values were normalized to the maximum intensity at pH 9.0 and emission was monitored at 510 nm. Absorbance readings around 280 nm are greatly altered due to the presence of DTT and thus are not shown.

FIG. 11. Fluorescence excitation spectra of rosGFP1 at various redox potentials. Fluorescence intensity values were normalized to the maximum intensity at E_o′-0.320 V and emission was monitored at 510 nm.

FIG. 12. Fluorescence excitation spectra of rosGFP3 at various redox potentials. The entire spectrum (A) shows the redox potential dependence on the excitation spectra of rosGFP3. Expanded the region around 405 nm (B), reveals the existence of an isosbestic point. Fluorescence intensity values were normalized to the maximum intensity at E_o′-0.330 V and emission was monitored at 510 nm.

FIG. 13. Fluorescence excitation spectra of rosGFP5 at various redox potentials. Fluorescence intensity values were normalized to the maximum intensity at E_o′-0.330 V and emission was monitored off the peak at 535 nm.

FIG. 14. A fluorescence excitation ratio results in the cancellation of pH artifacts. In the oxidized (A) or reduced (B) state, a ratio of fluorescence intensities at various excitation wavelengths of rosGFP2 is independent of pH.

FIG. 15. Dual-emission characteristics of rosGFP2. Excitation at 400 nm results in emission peaks centered near 450 and 510 nm, which have an opposite response to pH changes.

FIG. 16. A fluorescence emission ratio results in the cancellation of redox potential changes on pH determination. The fluorescence emission spectra (A) of rosGFP2 were collected at various redox potentials (ratios of DTT and DTT_ox) and at a constant pH of 6.0. Plotting the ratio of the two emission peaks results in a constant ratio over a large range of redox states (B). The dashed lines in B represent the maximum and minimum ratios to illustrate the possible dynamic range of rosGFP2 as a function of pH.

FIG. 17. A fluorescent micrograph showing the reticular localization pattern of rosGFP1 expressed in the mitochondrial matrix of an in vitro cultured HeLa cell, via fusion at the DNA level to the mitochondrial targeting sequence of the E₁α subunit of pyruvate dehydrogenase.

FIG. 18. Response of rosGFP1 to H₂O₂ and DTT induced redox potential changes in HeLa cell mitochondria. H₂O₂ and DTT were added at the indicted time points to a final concentration of 1 mM and 30 mM, respectively. The Fluorescence Intensity axis corresponds to the individual wavelengths, whereas the Ratio 400/480 axis corresponds to the ratio of the two wavelength channels.

FIG. 19. NADH-dependent reduction of rosGFP1 via lipoamide dehydrogenase. Each bar represents the percent reduction of oxidized rosGFP1 by 1-2 μL LDH, 1 mM lipoate, 1 mM NADH, and/or 1 mM DTT. Samples were equilibrated at 22° C. for one hour after which the fluorescence excitation was scanned from 325 to 525 nm. Percent reduction values were determined by the fluorescence at 490 nm with 100% corresponding to reduction by DTT.

FIG. 20. Fluorescence excitation spectra of rosGFP2 at varying concentrations of DTT_red and DTT_ox. Fluorescence emission intensity was monitored at 510 nm and normalized to the maximum intensity of the fully reduced spectrum (solid line).

FIG. 21. Redox equilibrium titration of rosGFP2 with dithiothreitol. The relative amount of reduced rosGFP2 at equilibrium (R) was measured using a ratio of the rosGFP2 fluorescence at 510 nm (excitation 490:425 nm). Oxidized rosGFP2 (1 μM) was incubated for four hours in 75 mM HEPES (pH 7.0), 140 mM NaCl, and 1 mM EDTA, containing varying ratios of DTT_red to DTT_ox (1 mM total). The equilibrium constant was determined by fitting the data according to equation 3. After nonlinear regression, a K_eq of 2.05×10^-2 was obtained (correlation coefficient: 0.998).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary stand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

• SEQ ID NO: 1 shows the amino acid sequence of wild-type GFP.
• SEQ ID NO: 2 shows the nucleic acid and amino acid sequence of rosGFP2.
• SEQ ID NO: 3 shows the amino acid sequence of rosGFP2.
• SEQ ID NO: 4 shows the nucleic acid and amino acid sequence of rosGFP1.
• SEQ ID NO: 5 shows the amino acid sequence of rosGFP1.
• SEQ ID NO: 6 shows the nucleic acid and amino acid sequence of rosGFP4.
• SEQ ID NO: 7 shows the amino acid sequence of rosGFP4.
• SEQ ID NO: 8 shows the nucleic acid and amino acid sequence of rosGFP3.
• SEQ ID NO: 9 shows the amino acid sequence of rosGFP3.
• SEQ ID NO: 10 shows the nucleic acid and amino acid sequence of rosGFP6.
• SEQ ID NO: 11 shows the amino acid sequence of rosGFP6.
• SEQ ID NO: 12 shows the nucleic acid and amino acid sequence of rosGFP5.
• SEQ ID NO: 13 shows the amino acid sequence of rosGFP5.
• SEQ ID NO: 14 shows the amino acid sequence of a tetrapeptide used to measure the ratio of thiol to disulfide in the cytosol and secretory pathway of cultured cells.
• SEQ ID NO: 15 shows the amino acid sequence of a nuclear localization sequence.
• SEQ ID NO: 16 shows the amino acid sequence of a mitochondrion localization sequence.
• SEQ ID NO: 17 shows the amino acid sequence of an endoplasmic reticulum localization sequence.

DETAILED DESCRIPTION

I. Abbreviations

	GFP	green fluorescent protein
	rosGFP	redox-sensitive GFP
	wtGFP	wild-type GFP

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Antibody: A polypeptide substantially encoded by an immunoglobulin gene or imunoglobulin genes, or fragments thereof, which specifically binds and recognizes an analyte (antigen). Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.

Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bind to GFP would be GFP-specific binding agents. Antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab′)2, a dimer of two Fab′ fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.

cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences. cDNA may also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is usually synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Conservative variations: Variants of a particular nucleic acid sequence, which encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified within a protein encoding sequence, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations," which are one species of conservative variations. Each nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation. The genetic code is shown in Table 1.

TABLE 1
First position	Second position	Third position

(5′ end)	U	C	A	G	(3′ end)
U	Phe	Ser	Tyr	Cys	U
	Phe	Ser	Tyr	Cys	C
	Leu	Ser	Stop	Stop	A
	Leu	Ser	Stop	Trp	G
C	Leu	Pro	His	Arg	U
	Leu	Pro	His	Arg	C
	Leu	Pro	Gln	Arg	A
	Leu	Pro	Gln	Arg	G
A	Ile	Thr	Asn	Ser	U
	Ile	Thr	Asn	Ser	C
	Ile	Thr	Lys	Arg	A
	Met	Thr	Lys	Arg	G
G	Val	Ala	Asp	Gly	U
	Val	Ala	Asp	Gly	C
	Val	Ala	Glu	Gly	A
	Val	Ala	Glu	Gly	G

One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

Furthermore, one of ordinary skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitutions providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

• 1) Alanine (A), Serine (S), Threonine (T);
• 2) Aspartic acid (D), Glutamic acid (E);
• 3) Asparagine (N), Glutaminc (Q);
• 4) Arginine (R), Lysine (K);
• 5) Isoleucine (I), Leucine (L), Methionine (M), Valime (V); and
• 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  Not all residue positions within a protein will tolerate an otherwise "conservative" substitution. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. By way of example, in a GFP the residues that compose the chromophore do not generally tolerate amino acid substitutions.

Epitope tags: Short stretches of amino acids to which a specific antibody can be raised, which in some embodiments allows one to specifically identify and track the tagged protein that has been added to a living organism or to cultured cells. Detection of the tagged molecule can be achieved using a number of different techniques. Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("western"), and affinity chromatography. Examples of useful epitope tags include FLAG, T7, HA (hemagglutinin) and myc.

Expression control sequence: This phrase refers to nucleotide sequences that regulate the expression of a nucleotide sequence to which they are operatively linked. Expression control sequences are "operatively linked" to a nucleotide sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleotide sequence. Thus, expression control sequence(s) can include promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding sequence, intron splicing signals, and stop codons.

Fluorescent property: A characteristic of a fluorescent molecule. Examples of fluorescent properties include the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum (the "fluorescence spectrum", the excitation wavelength maximum and 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, or the fluorescence anisotropy. A measurable difference in any one of these properties between wild-type Aequorea GFP and the mutant form is useful. A measurable difference can be determined by determining the amount of any quantitative fluorescent property, e.g., the amount of fluorescence at a particular wavelength, or the integral of fluorescence over the emission spectrum. Determining ratios of excitation amplitude or emission amplitude at two different wavelengths ("excitation amplitude ratioing" and "emission amplitude ratioing," respectively) for a particular molecule are advantageous. The ratioing process provides an internal reference and cancels out variations, for instance in the absolute brightness of the excitation source, the sensitivity of the detector, and light scattering or quenching by the sample.

Fusion protein: Proteins that have two (or more) parts fused together, which are not found joined together in nature. In general, the two domains are genetically fused together, in that nucleic acid molecules that encode each protein domain are functionally linked together, for instance by a linker oligonucleotide, thereby producing a single fusion-encoding nucleic acid molecule. The translated product of such a fusion-encoding nucleic acid molecule is the fusion protein.

Green fluorescent protein (GFP): GFP is a 238 amino acid, spontaneously fluorescent protein, originally isolated from the Pacific Northwest jellyfish Aequorea victoria. The amino acid sequence of wtGFP is shown in SEQ ID NO: 1. Tis protein has become an extremely popular tool in molecular and call biology (for reviews: Tsien, Annu. Rev. Biochem. 67:509-544, 1998; Remington, In Bioluminescence and chemiluminescence (eds. T. O. Baldwin and M. M. Sigler), pp. 195-211, 2000, Academic, San Diego, Calif.). Originally GFP was used as a passive indicator of gene expression and protein localization. More recently, GFP has taken on the role of an active indicator of such things as intracellular H⁺, Ca²⁺, and halide ion concentrations (Kneen et al., Biophys. J. 74:1591-1599, 1998; Llopis et al., Proc. Natl. Acad. Sci. USA 95:6803-6808, 1998; Baird et al., Proc. Natl. Acad. Sci. USA 96:11241-11246, 1999; Jayaraman et al., J. Biol. Chem. 275:6047-6050, 2000).

In addition to GFP being highly fluorescent, protease resistant, and very stable throughout a wide range of pH and solvent conditions, it also has the advantage of being functional as a single protein encoded by a single gene. These traits result in a biological probe molecule that can be expressed in nearly all organisms. It also can be targeted to subcellular organelles by a host cell, for instance through the inclusion of a targeting sequence on the construction from which it is expressed. GFP is a non-invasive indicator, which allows for experiments to be conducted and monitored in a single cell over a period of time.

GFPs as discussed herein (including rosGFPs) can be expressed as fusion proteins. The GFP protein can be functionally fused to, for instance, a tag (such as an epitope tag), a targeting molecule (such as a targeting peptide), or a protein (or fragment thereof) that provides an additional function, such as a biochemical, biological, or localization function. The construction and production of fusion proteins is well known to one of ordinary skill in the art.

A "mutant" GFP is a green fluorescent protein (or nucleic acid encoding such) that has at least one residue that is different from (mutated from) the wtGFP. Mutations include, for instance, conservative or non-conservative amino acid substitutions, silent mutations (wherein the nucleic acid sequence is different from wild-type at a particular residue, but the amino acid sequence is not), insertions (including fusion proteins), and deletions. Myriad mutant GFPs are known, including for instance those disclosed in the following patent documents: U.S. Pat. Nos. 5,804,387; 6,090,919; 6,096,865; 6,054,321; 5,625,049; 5,874,304; 5,777,078; 5,968,750; 6,020,192; and 6,146,826; and published international patent application WO 99/64592.

Specific examples of mutant GFPs include proteins in which the fluorescence spectrum of the mutant is responsive to an environmental variable, such as temperature, proton concentration (pH), salt concentration, and redox status. Particular mutant GFPs as provided herein are sensitive to redox status, and others are responsive to both redox status and pH. A fluorescence spectrum is "responsive" to an environmental variable if the spectrum changes with changes in that variable.

Immunoassay: An assay that utilizes an antibody to specifically bind an analyte. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, detect, and/or quantify the analyte, or alternately using a particularly analyte (e.g., an antigen) to isolate, target, detect, and/or quantify the antibody.

In vitro amplification: Techniques that increases the number of copies of a nucleic acid molecule in a sample or specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques. Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Pat. No. 5,744,311); transcription-free isothermal amplification (see U.S. Pat. No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Pat. No. 6,025,134).

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Label: A composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P (or other radio-isotope), fluorescent dyes, fluorescent proteins, electron-dense reagents, enzymes (e.g., for use in an ELISA), biotin, dioxigenin, or haptens and proteins or peptides for which antisera or monoclonal antibodies are available. A label often generates a measurable signal, such as radioactivity, fluorescent light or enzyme activity, which can be used to detect and/or quantitate the amount of labeled molecule.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form. Unless otherwise limited, this term encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. When a nucleic acid molecule is represented herein by a DNA sequence, the corresponding RNA molecules are likewise understood, in which "U" replaces "T."

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occu