- Email: [email protected]
Förster Distances between Green Fluorescent Protein Pairs
438
NOTES & TIPS
10. Moran, J. V., Holmes, S. E., Naas, T. P., DeBerardinis, R. J., Boeke, J. D., and Kazazian, H. H. (1996) High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927. 11. Freeman, J. D., Goodchild, N. L., and Mager, D. L. (1994) A modified indicator gene for selection of retrotransposition events in mammalian cells. BioTechniques 17, 47–52. 12. Naas, T. P., DeBerardinis, R. J., Moran, J. V., Ostertag, E. M., Kingsmore, S. F., Seldin, M. F., Hayashizaki, Y., Martin, S. L., and Kazazian, H. H. (1998) An actively retrotransposing, novel subfamily of mouse L1 elements. EMBO J. 17, 590 –597. 13. Ostertag, E. M., Prak, E. T., DeBerardinis, R. J., Moran, J. V., and Kazazian, H. H. (2000) Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Res. 28, 1418 –1423. 14. Kimberland, M. L., Divoky, V., Prchal, J., Schwahn, U., Berger, W., and Kazazian, H. H. (1999) Full-length human L1 insertions retain the capacity for high frequency retrotransposition in cultured cells. Hum. Mol. Genet. 8, 1557–1560. 15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Fo¨rster Distances between Green Fluorescent Protein Pairs George H. Patterson,* David W. Piston,* and B. George Barisas† ,1 *Department of Biophysics, Vanderbilt University, Nashville, Tennessee 37232; and †Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 Received May 18, 2000
Since its initial description by Fo¨rster in 1948 (1), fluorescence resonant energy transfer (FRET) 2 has proved an invaluable tool for cell biology by permitting assessment of nanometer proximity between specific pairs of cellular proteins. For many years, such protein pairs were limited to species which could be labeled by antibodies, hormone, or other ligands conjugated with suitable pairs of dyes. However, the cloning of green fluorescent protein (GFP) in 1992 (2) has been followed by introduction of GFP spectral variants, including enhanced yellow, green, cyan, and blue fluorescent proteins and, most recently, a red fluorescent coral protein DsRed (3), now available commercially (Clontech Lab1 To whom correspondence should be addressed. Fax: (970) 4911801. E-mail: [email protected]. 2 Abbreviations used: DsRed, red fluorescent protein from Discosoma; EBFP, enhanced blue fluorescent protein (F64L/S65T/Y66H/ Y145F); ECFP, enhanced cyan fluorescent protein (F64L/S65T/ Y66W/N146I/M153T/V163A); EGFP, enhanced green fluorescent protein (F64L/S65T); EYFP, enhanced yellow fluorescent protein (S65G/V68L/S72A/T203Y); FRET, fluorescence resonant energy transfer; GFP, green fluorescent protein (used here to also include the DsRed coral protein).
Analytical Biochemistry 284, 438 – 440 (2000) doi:10.1006/abio.2000.4708 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
oratories, Palo Alto, CA). These materials have permitted genetic introduction into cells of intrinsically fluorescent protein constructs which frequently possess the biological functionality of the parent protein. These chromophores thus permit interprotein energy transfer to be observed without application of extrinsic fluorophores. Such observations have proved extremely popular (see, for example, 4), so that in early 2000, the MedLine index listed over 50 articles containing FRET observations of GFP species. Unfortunately, most such data have been interpreted only qualitatively assuming that appreciable energy transfer implies an interprotein distance of less than, say, 100 Å, but the actual Fo¨rster distances r 0 for pairs of GFP species have apparently not been calculated. Our laboratories have been involved both in photophysical characterization of GFP luminescence (5) and in cell biological studies involving these proteins (6). We thus have calculated Fo¨rster distances for fluorescence resonant energy transfer between the major color variants of green fluorescent protein and the coral protein DsRed and wish to make these results available to other workers. These values allow interpretation of fluorescence resonant energy transfer efficiencies in terms of actual interchromophore distances. Materials and Methods GFP expression and purification. Plasmids for Histagged green fluorescent protein color variants were prepared as previously described (5) and transformed into the Escherichia coli strain BL21 pLysS for protein expression. The His-tagged GFP variant proteins were grown in E. coli as previously described (5). After inoculation, cultures were incubated for 2 h, induced with 0.1 mM isopropyl beta;-D-thiogalactopyranoside, and grown for 5 h before harvesting by centrifugation. Supernatant from sonicated cells was passed over Ni– NTA, the column washed, and GFP eluted by an imidazole-containing buffer. Protein purity (⬎95%) was assessed by scanning densitometry of sodium dodecyl sulfate gels stained with Coomassie brilliant blue. Spectroscopy. Extinction coefficients were calculated using Beer’s law and the absorbance of 7 M, pH 8.0 protein solutions measured as previously described (5) in a Hewlett–Packard 8453 UV–visible spectrophotometer (Waldbronn, Germany). Fluorescence excitation and emission measurements were performed on a SPEX 1681 Fluorolog spectrofluorometer (Edison, NJ). Quantum yield measurements were performed as described (5), using equal 488-nm optical densities of each variant at pH 8.0 and 1-aminoanthracene (quantum yield 0.61) or fluorescein (quantum yield 0.85) reference standards for EBFP and other GFPs, respectively. Absorption and fluorescence emission spectra of DsRed were supplied by the proteins’ investigators (3).
439
NOTES & TIPS
FIG. 1. Spectral overlap integral for fluorescence resonant energy transfer from EBFP donor to EGFP acceptor. Donor fluorescence emission and acceptor absorption are plotted vs wavelength together with the integrand, namely, their product multiplied by 4. It is apparent that the integrand closely approaches zero at both the upper and lower wavelength limits of integration.
Calculations. Fo¨rster’s theory (1) shows that the rate constant for singlet energy transfer between chromophores varies with the inverse sixth power of the interchromophore distance r. The efficiency E of energy transfer between chromophores is thus [1 ⫹ (r/r 0 ) 6 ] ⫺1 so that the critical distance r 0 for energy transfer is that distance at which E is 50%. The equation which defines r 0 in terms of spectral properties of chromophore pairs, rarely seen in its full form, can be derived from Fo¨rster (1): r 06 ⫽
2303 2 9 Dn ⫺4 4共2 兲 5 N
冕
⬁
f共 兲 ⑀ 共 兲 4 d ,
0
where c is the speed of light, 2303 is ln10 ⫻ 1000 cm 3 L ⫺1, N is Avogadro’s number, is the factor reflecting relative orientation of the chromophore transition dipoles and equaling 2/3 for rapid random orientation, D is the overall quantum yield for donor fluorescence, and n is the index of refraction of the medium separat-
ing the chromophores. In the most convenient choice of units, the constants preceding have an aggregate value of 8.786 ⫻ 10 ⫺11 mol L ⫺1 cm nm 2. Within the integral, i.e., the spectral overlap integral J, f( ) is the normalized fluorescence emission intensity, i.e., its integral over wavelength is unity, and ⑀() is the acceptor molar absorptivity. For ⑀() given in the usual units, the integral has dimensions of L mol ⫺1 cm ⫺1 nm 4. Overlap integrals were calculated by numerical integration of the normalized fluorescence emission and absorption spectra (Fig. 1) for various pairs of GFP derivatives. In all cases, the integrand closely approached zero at both the upper and lower wavelength limits of integration. In turn, Fo¨rster distances were calculated assuming rapid randomization of relative fluorophore orientation, i.e., ⫽ 2/3, and an index of refraction of 1.3342, namely, the value for water at 25 deg. Measurements of GFP molar absorptivity have a relative standard deviation of approximately 8% when replicated using independent protein preparations. Assuming an equal, independent uncertainty in the fluorescence quantum yield, the standard errors of the r 0 values are conservatively estimated through propagation of error methods (7) at about 2%. Results and Discussion Fo¨rster distances (in nanometers) for energy transfer between various combinations of enhanced green fluorescent protein variants are presented in Table 1. For donor–acceptor pairs where the acceptor absorption maximum lies to the red of the donor emission peak, these distances range from 3.17 (EBFP–DsRed) to 5.64 nm (EGFP–EYFP). Examination of a comprehensive compilation of Fo¨rster energy transfer distances by Wu and Brand (8) shows that the 5.64-nm r 0 calculated for EGFP–EYFP is one of the larger r 0 values encountered to date. For example, r 0 values of 4.9 –5.4 nm have been reported for fluorescein isothiocyanate and tetramethylrhodamine (9, 10) and this pair of chromophores is often considered the standard for high energy transfer efficiency. While EGFP–EYFP
TABLE 1
Fo¨rster Distances (nm) for Energy Transfer between Various Combinations of Enhanced GFP Variants or DsRed Acceptor (enhanced GFP variant or DsRed) Donor
Blue
Cyan
Green
Yellow
Red
Blue Cyan Green Yellow Red
2.61 ⫾ 0.05 a — — — —
3.77 ⫾ 0.08 3.28 ⫾ 0.07 1.93 ⫾ 0.04 1.00 ⫾ 0.02 1.40 ⫾ 0.03
4.14 ⫾ 0.08 4.82 ⫾ 0.10 4.65 ⫾ 0.09 3.25 ⫾ 0.07 2.84 ⫾ 0.06
3.82 ⫾ 0.08 4.92 ⫾ 0.10 5.64 ⫾ 0.11 5.11 ⫾ 0.10 3.14 ⫾ 0.06
3.17 ⫾ 0.06 4.17 ⫾ 0.08 4.73 ⫾ 0.09 4.94 ⫾ 0.10 3.54 ⫾ 0.07
a r 0 values are given in nanometers. Uncertainties indicated for these quantities are the estimated standard deviations of r 0 as calculated by propagation of error methods (7).
440
NOTES & TIPS
exhibits the strongest FRET of any combination of GFPs, r 0 for the pair ECFP–EYFP is only slightly smaller at 4.90 nm. This combination of proteins is particularly well suited for applications where acceptor is photobleached in the presence of donor and the enhancement of donor fluorescence recorded (11). Since EYFP exhibits substantial absorbance at 540 nm, well above the long-wavelength limit of ECFP absorption, the yellow acceptor protein can be effectively bleached with little damage to the cyan donor. DsRed is seen to be an effective acceptor for EGFP and EYFP fluorescence, r 0 values being 4.73 and 4.94 nm, respectively. The long-wavelength tail of this protein’s red fluorescence should particularly facilitate detection of sensitized emission from green or yellow donors. Energy transfer occurs between all pairs of chromophores whose spectra overlap and we include r 0 values for several combinations where the donor emission peaks to the red of acceptor absorption. Because the GFP variants exhibit only moderate Stokes shifts, such r 0 values can be substantial. Thus energy transfer from EYFP to EGFP would be expected to exhibit a Fo¨rster distance of 3.25 nm. Finally, since excitation transfer from one chromophore to another of the same kind can depolarize fluorescence emission, we include these values as well. For example, transfer from one EYFP to another should exhibit an r 0 of 5.11 nm, a substantial value again arising from relatively small Stokes shifts. The validity of energy transfer distances calculated for this method is well documented. Fo¨rster (12) demonstrated the concentration dependence of energy transfer in dye solutions while Stryer and Haugland (13) measured transfer efficiency between donor–acceptor pairs physically separated by specific distances. In all such cases, the r 0 values calculated from spectroscopic data have agreed well with direct FRET measurements. The much larger uncertainty in predicting energy transfer distances involves relative chromophore orientation. We have followed the common practice of assuming that donor and acceptor undergo orientational randomization which is rapid with respect to donor lifetime, namely, that is 2/3. While this can be strictly justified only for independent molecules in solution, if information about actual chromophore orientation is available, then r 0 values can be easily corrected to reflect this.
Acknowledgments. The authors are grateful to Dr. Aleksandr P. Savitsky of the Institute of Biochemistry, Russian Academy of Science, Moscow, for providing spectral information on DsRed. This work was supported in part by NIH Grants AI36306 (B.G.B.) and DK53434 (D.W.P.) and by NSF Grants MCB-9807822 (B.G.B.) and DBI-9871063 (D.W.P.). During part of this work G.H.P. was an NIH Trainee in Molecular Biophysics (GM08320).
REFERENCES 1. Fo¨rster, V. T. (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann. Phys. 6, 54 –75. 2. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229 –233. 3. Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Markelov, M. L., and Lukyanov, S. A. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969 –973. 4. Sako, Y., Minoghchi, S., and Yanagida, T. (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168 –172. 5. Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., and Piston, D. W. (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790. 6. Nelson, S., Horvat, R. D., Malvey, J., Roess, D. A., Barisas, B. G., and Clay, C. M. (1999) Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion. Endocrinology 140, 950 –957. 7. Bevington, P. R. (1969) Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York. 8. Wu, P., and Brand, L. (1994) Resonance energy transfer: Methods and applications. Anal. Biochem. 218, 1–13. 9. Johnson, D. A., Voet, J. G., and Taylor, P. (1984) Fluorescence energy transfer between cobra ␣-toxin molecules bound to the acetylcholine receptor. J. Biol. Chem. 259, 5717–5725. 10. Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) Fluorescence energy transfer studies of purified erythrocyte Ca 2⫹ATPase. Ca 2⫹-regulated activation by oligomerization. J. Biol. Chem. 264, 19495–19499. 11. Kenworthy, A. K., and Edidin, M. (1998) Distribution of glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of ⬍100 Å using imaging fluorescence resonance energy transfer. J. Cell Biol. 142, 69 – 84. ¨ ber12. Fo¨rster, T. H. (1948) Versuche zum zwischenmolekularen U gang von elektronenanregungsenergie. Naturwissenschaften 33, 93–100. 13. Stryer, L., and Haugland, R. P. (1967) Energy transfer: A spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719 –726.
NOTES & TIPS
10. Moran, J. V., Holmes, S. E., Naas, T. P., DeBerardinis, R. J., Boeke, J. D., and Kazazian, H. H. (1996) High frequency retrotransposition in cultured mammalian cells. Cell 87, 917–927. 11. Freeman, J. D., Goodchild, N. L., and Mager, D. L. (1994) A modified indicator gene for selection of retrotransposition events in mammalian cells. BioTechniques 17, 47–52. 12. Naas, T. P., DeBerardinis, R. J., Moran, J. V., Ostertag, E. M., Kingsmore, S. F., Seldin, M. F., Hayashizaki, Y., Martin, S. L., and Kazazian, H. H. (1998) An actively retrotransposing, novel subfamily of mouse L1 elements. EMBO J. 17, 590 –597. 13. Ostertag, E. M., Prak, E. T., DeBerardinis, R. J., Moran, J. V., and Kazazian, H. H. (2000) Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Res. 28, 1418 –1423. 14. Kimberland, M. L., Divoky, V., Prchal, J., Schwahn, U., Berger, W., and Kazazian, H. H. (1999) Full-length human L1 insertions retain the capacity for high frequency retrotransposition in cultured cells. Hum. Mol. Genet. 8, 1557–1560. 15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Fo¨rster Distances between Green Fluorescent Protein Pairs George H. Patterson,* David W. Piston,* and B. George Barisas† ,1 *Department of Biophysics, Vanderbilt University, Nashville, Tennessee 37232; and †Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 Received May 18, 2000
Since its initial description by Fo¨rster in 1948 (1), fluorescence resonant energy transfer (FRET) 2 has proved an invaluable tool for cell biology by permitting assessment of nanometer proximity between specific pairs of cellular proteins. For many years, such protein pairs were limited to species which could be labeled by antibodies, hormone, or other ligands conjugated with suitable pairs of dyes. However, the cloning of green fluorescent protein (GFP) in 1992 (2) has been followed by introduction of GFP spectral variants, including enhanced yellow, green, cyan, and blue fluorescent proteins and, most recently, a red fluorescent coral protein DsRed (3), now available commercially (Clontech Lab1 To whom correspondence should be addressed. Fax: (970) 4911801. E-mail: [email protected]. 2 Abbreviations used: DsRed, red fluorescent protein from Discosoma; EBFP, enhanced blue fluorescent protein (F64L/S65T/Y66H/ Y145F); ECFP, enhanced cyan fluorescent protein (F64L/S65T/ Y66W/N146I/M153T/V163A); EGFP, enhanced green fluorescent protein (F64L/S65T); EYFP, enhanced yellow fluorescent protein (S65G/V68L/S72A/T203Y); FRET, fluorescence resonant energy transfer; GFP, green fluorescent protein (used here to also include the DsRed coral protein).
Analytical Biochemistry 284, 438 – 440 (2000) doi:10.1006/abio.2000.4708 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
oratories, Palo Alto, CA). These materials have permitted genetic introduction into cells of intrinsically fluorescent protein constructs which frequently possess the biological functionality of the parent protein. These chromophores thus permit interprotein energy transfer to be observed without application of extrinsic fluorophores. Such observations have proved extremely popular (see, for example, 4), so that in early 2000, the MedLine index listed over 50 articles containing FRET observations of GFP species. Unfortunately, most such data have been interpreted only qualitatively assuming that appreciable energy transfer implies an interprotein distance of less than, say, 100 Å, but the actual Fo¨rster distances r 0 for pairs of GFP species have apparently not been calculated. Our laboratories have been involved both in photophysical characterization of GFP luminescence (5) and in cell biological studies involving these proteins (6). We thus have calculated Fo¨rster distances for fluorescence resonant energy transfer between the major color variants of green fluorescent protein and the coral protein DsRed and wish to make these results available to other workers. These values allow interpretation of fluorescence resonant energy transfer efficiencies in terms of actual interchromophore distances. Materials and Methods GFP expression and purification. Plasmids for Histagged green fluorescent protein color variants were prepared as previously described (5) and transformed into the Escherichia coli strain BL21 pLysS for protein expression. The His-tagged GFP variant proteins were grown in E. coli as previously described (5). After inoculation, cultures were incubated for 2 h, induced with 0.1 mM isopropyl beta;-D-thiogalactopyranoside, and grown for 5 h before harvesting by centrifugation. Supernatant from sonicated cells was passed over Ni– NTA, the column washed, and GFP eluted by an imidazole-containing buffer. Protein purity (⬎95%) was assessed by scanning densitometry of sodium dodecyl sulfate gels stained with Coomassie brilliant blue. Spectroscopy. Extinction coefficients were calculated using Beer’s law and the absorbance of 7 M, pH 8.0 protein solutions measured as previously described (5) in a Hewlett–Packard 8453 UV–visible spectrophotometer (Waldbronn, Germany). Fluorescence excitation and emission measurements were performed on a SPEX 1681 Fluorolog spectrofluorometer (Edison, NJ). Quantum yield measurements were performed as described (5), using equal 488-nm optical densities of each variant at pH 8.0 and 1-aminoanthracene (quantum yield 0.61) or fluorescein (quantum yield 0.85) reference standards for EBFP and other GFPs, respectively. Absorption and fluorescence emission spectra of DsRed were supplied by the proteins’ investigators (3).
439
NOTES & TIPS
FIG. 1. Spectral overlap integral for fluorescence resonant energy transfer from EBFP donor to EGFP acceptor. Donor fluorescence emission and acceptor absorption are plotted vs wavelength together with the integrand, namely, their product multiplied by 4. It is apparent that the integrand closely approaches zero at both the upper and lower wavelength limits of integration.
Calculations. Fo¨rster’s theory (1) shows that the rate constant for singlet energy transfer between chromophores varies with the inverse sixth power of the interchromophore distance r. The efficiency E of energy transfer between chromophores is thus [1 ⫹ (r/r 0 ) 6 ] ⫺1 so that the critical distance r 0 for energy transfer is that distance at which E is 50%. The equation which defines r 0 in terms of spectral properties of chromophore pairs, rarely seen in its full form, can be derived from Fo¨rster (1): r 06 ⫽
2303 2 9 Dn ⫺4 4共2 兲 5 N
冕
⬁
f共 兲 ⑀ 共 兲 4 d ,
0
where c is the speed of light, 2303 is ln10 ⫻ 1000 cm 3 L ⫺1, N is Avogadro’s number, is the factor reflecting relative orientation of the chromophore transition dipoles and equaling 2/3 for rapid random orientation, D is the overall quantum yield for donor fluorescence, and n is the index of refraction of the medium separat-
ing the chromophores. In the most convenient choice of units, the constants preceding have an aggregate value of 8.786 ⫻ 10 ⫺11 mol L ⫺1 cm nm 2. Within the integral, i.e., the spectral overlap integral J, f( ) is the normalized fluorescence emission intensity, i.e., its integral over wavelength is unity, and ⑀() is the acceptor molar absorptivity. For ⑀() given in the usual units, the integral has dimensions of L mol ⫺1 cm ⫺1 nm 4. Overlap integrals were calculated by numerical integration of the normalized fluorescence emission and absorption spectra (Fig. 1) for various pairs of GFP derivatives. In all cases, the integrand closely approached zero at both the upper and lower wavelength limits of integration. In turn, Fo¨rster distances were calculated assuming rapid randomization of relative fluorophore orientation, i.e., ⫽ 2/3, and an index of refraction of 1.3342, namely, the value for water at 25 deg. Measurements of GFP molar absorptivity have a relative standard deviation of approximately 8% when replicated using independent protein preparations. Assuming an equal, independent uncertainty in the fluorescence quantum yield, the standard errors of the r 0 values are conservatively estimated through propagation of error methods (7) at about 2%. Results and Discussion Fo¨rster distances (in nanometers) for energy transfer between various combinations of enhanced green fluorescent protein variants are presented in Table 1. For donor–acceptor pairs where the acceptor absorption maximum lies to the red of the donor emission peak, these distances range from 3.17 (EBFP–DsRed) to 5.64 nm (EGFP–EYFP). Examination of a comprehensive compilation of Fo¨rster energy transfer distances by Wu and Brand (8) shows that the 5.64-nm r 0 calculated for EGFP–EYFP is one of the larger r 0 values encountered to date. For example, r 0 values of 4.9 –5.4 nm have been reported for fluorescein isothiocyanate and tetramethylrhodamine (9, 10) and this pair of chromophores is often considered the standard for high energy transfer efficiency. While EGFP–EYFP
TABLE 1
Fo¨rster Distances (nm) for Energy Transfer between Various Combinations of Enhanced GFP Variants or DsRed Acceptor (enhanced GFP variant or DsRed) Donor
Blue
Cyan
Green
Yellow
Red
Blue Cyan Green Yellow Red
2.61 ⫾ 0.05 a — — — —
3.77 ⫾ 0.08 3.28 ⫾ 0.07 1.93 ⫾ 0.04 1.00 ⫾ 0.02 1.40 ⫾ 0.03
4.14 ⫾ 0.08 4.82 ⫾ 0.10 4.65 ⫾ 0.09 3.25 ⫾ 0.07 2.84 ⫾ 0.06
3.82 ⫾ 0.08 4.92 ⫾ 0.10 5.64 ⫾ 0.11 5.11 ⫾ 0.10 3.14 ⫾ 0.06
3.17 ⫾ 0.06 4.17 ⫾ 0.08 4.73 ⫾ 0.09 4.94 ⫾ 0.10 3.54 ⫾ 0.07
a r 0 values are given in nanometers. Uncertainties indicated for these quantities are the estimated standard deviations of r 0 as calculated by propagation of error methods (7).
440
NOTES & TIPS
exhibits the strongest FRET of any combination of GFPs, r 0 for the pair ECFP–EYFP is only slightly smaller at 4.90 nm. This combination of proteins is particularly well suited for applications where acceptor is photobleached in the presence of donor and the enhancement of donor fluorescence recorded (11). Since EYFP exhibits substantial absorbance at 540 nm, well above the long-wavelength limit of ECFP absorption, the yellow acceptor protein can be effectively bleached with little damage to the cyan donor. DsRed is seen to be an effective acceptor for EGFP and EYFP fluorescence, r 0 values being 4.73 and 4.94 nm, respectively. The long-wavelength tail of this protein’s red fluorescence should particularly facilitate detection of sensitized emission from green or yellow donors. Energy transfer occurs between all pairs of chromophores whose spectra overlap and we include r 0 values for several combinations where the donor emission peaks to the red of acceptor absorption. Because the GFP variants exhibit only moderate Stokes shifts, such r 0 values can be substantial. Thus energy transfer from EYFP to EGFP would be expected to exhibit a Fo¨rster distance of 3.25 nm. Finally, since excitation transfer from one chromophore to another of the same kind can depolarize fluorescence emission, we include these values as well. For example, transfer from one EYFP to another should exhibit an r 0 of 5.11 nm, a substantial value again arising from relatively small Stokes shifts. The validity of energy transfer distances calculated for this method is well documented. Fo¨rster (12) demonstrated the concentration dependence of energy transfer in dye solutions while Stryer and Haugland (13) measured transfer efficiency between donor–acceptor pairs physically separated by specific distances. In all such cases, the r 0 values calculated from spectroscopic data have agreed well with direct FRET measurements. The much larger uncertainty in predicting energy transfer distances involves relative chromophore orientation. We have followed the common practice of assuming that donor and acceptor undergo orientational randomization which is rapid with respect to donor lifetime, namely, that is 2/3. While this can be strictly justified only for independent molecules in solution, if information about actual chromophore orientation is available, then r 0 values can be easily corrected to reflect this.
Acknowledgments. The authors are grateful to Dr. Aleksandr P. Savitsky of the Institute of Biochemistry, Russian Academy of Science, Moscow, for providing spectral information on DsRed. This work was supported in part by NIH Grants AI36306 (B.G.B.) and DK53434 (D.W.P.) and by NSF Grants MCB-9807822 (B.G.B.) and DBI-9871063 (D.W.P.). During part of this work G.H.P. was an NIH Trainee in Molecular Biophysics (GM08320).
REFERENCES 1. Fo¨rster, V. T. (1948) Zwischenmolekulare energiewanderung und fluoreszenz. Ann. Phys. 6, 54 –75. 2. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229 –233. 3. Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Markelov, M. L., and Lukyanov, S. A. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969 –973. 4. Sako, Y., Minoghchi, S., and Yanagida, T. (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168 –172. 5. Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., and Piston, D. W. (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790. 6. Nelson, S., Horvat, R. D., Malvey, J., Roess, D. A., Barisas, B. G., and Clay, C. M. (1999) Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion. Endocrinology 140, 950 –957. 7. Bevington, P. R. (1969) Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York. 8. Wu, P., and Brand, L. (1994) Resonance energy transfer: Methods and applications. Anal. Biochem. 218, 1–13. 9. Johnson, D. A., Voet, J. G., and Taylor, P. (1984) Fluorescence energy transfer between cobra ␣-toxin molecules bound to the acetylcholine receptor. J. Biol. Chem. 259, 5717–5725. 10. Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) Fluorescence energy transfer studies of purified erythrocyte Ca 2⫹ATPase. Ca 2⫹-regulated activation by oligomerization. J. Biol. Chem. 264, 19495–19499. 11. Kenworthy, A. K., and Edidin, M. (1998) Distribution of glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of ⬍100 Å using imaging fluorescence resonance energy transfer. J. Cell Biol. 142, 69 – 84. ¨ ber12. Fo¨rster, T. H. (1948) Versuche zum zwischenmolekularen U gang von elektronenanregungsenergie. Naturwissenschaften 33, 93–100. 13. Stryer, L., and Haugland, R. P. (1967) Energy transfer: A spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719 –726.