Abstract
The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.
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REFERENCES
Tsien, R. Y. (1998) Annu. Rev. Biochem., 67, 509–544.
Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G., and Ward, W. W. (1993) Biochemistry, 32, 1212–1218.
Niwa, G., Inouye, S., Hirano, T., Matsuno, T., Kojima, S., Massayuki, K., Ohashi, M., and Tsuji, F. I. (1996) Proc. Natl. Acad. Sci. USA, 93, 13617–13622.
Yang, F., Moss, L. G., and Phillips, G. N. (1996) Nature Biotechnol., 14, 1246–1251.
Phillips, G. N., Jr. (1998) in Green Fluorescent Protein: Properties, Applications, and Protocols (Chalfie, M., and Kain, R., eds.) Wiley-Liss, New York, pp. 77–96.
Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., and Lukyanov, S. A. (1999) Nat. Biotechnol., 17, 969–973.
Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K., and Tsien, R. Y. (2000) Proc. Natl Acad. Sci. USA, 97, 11990–11995.
Wall, M. A., Socolich, M., and Ranganathan, R. (2000) Nature Struct. Biol., 7, 1133–1138.
Reid, B. G., and Flynn, G. C. (1997) Biochemistry, 36, 6789–6791.
Vrzheshch, P. V., Akovbian, N. A., Varfolomeyev, S. D., and Verkhusha, V. V. (2000) FEBS Lett., 487, 203–208.
Baird, S. G., Zacharias, D. A., and Tsien, R. Y. (2000) Proc. Natl. Acad. Sci. USA, 97, 11984–11989.
Bokman, S. H., and Ward, W. W. (1981) Biochem. Biophys. Res. Commun., 101, 1372–1380.
Ward, W. W. (1981) in Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications (DeLuca, M., and McElroy, D. W., eds.) Academic Press, New York, pp. 235–242.
Llopis, J., McCaffery, J. M., Miyawaki, A., Farquhar, M. G., and Tsien, R. Y. (1998) Proc. Natl. Acad. Sci. USA, 95, 6803–6808.
Elsliger, M., Wachter, R. M., Hanson, G. T., Kallio, K., and Remington, S. J. (1999) Biochemistry, 38, 5296–5301.
Ward, W. W. (1998) in Green Fluorescent Protein: Properties, Applications, and Protocols (Chalfie, M., and Kain, R., eds.) Wiley-Liss, New York, pp. 45–75.
Freifelder, D. (1976) Physical Biochemistry, W. H. Freeman and Company, San Francisco.
Wiedenmann, J., Elke, C., Spindler, K. D., and Funke, W. (2000) Proc. Natl. Acad. Sci. USA, 97, 14091–14096.
Culter, M. W., and Ward, W. W. (1997) in Bioluminescence and Chemiluminescence: Molecular Reporting with Photons (Hastings, J. W., Kricka, L. J., and Stanley, P. E., eds.) Wiley, New York, pp. 403–406.
Yarbrough, D., Wachter, R. M., Kallio, K., Matz, M. V., and Remington, S. J. (2001) Proc. Natl. Acad. Sci. USA, 98, 462–467.
Varfolomeyev, S. D., and Gurevich, K. G. (1999) in Biokinetics: Practical Course [in Russian], FAIR-PRESS Moscow.
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Verkhusha, V.V., Akovbian, N.A., Efremenko, E.N. et al. Kinetic Analysis of Maturation and Denaturation of DsRed, a Coral-Derived Red Fluorescent Protein. Biochemistry (Moscow) 66, 1342–1351 (2001). https://doi.org/10.1023/A:1013325627378
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DOI: https://doi.org/10.1023/A:1013325627378