Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Jun;74(6):3165–3172. doi: 10.1016/S0006-3495(98)78022-1

Tyrosine quenching of tryptophan phosphorescence in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

G B Strambini 1, E Gabellieri 1, M Gonnelli 1, S Rahuel-Clermont 1, G Branlant 1
PMCID: PMC1299656  PMID: 9635769

Abstract

Tyrosine is known to quench the phosphorescence of free tryptophan derivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demonstrated. This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by comparing the phosphorescence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, is replaced with phenylalanine or Y283 is replaced by valine. Phosphorescence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp84-->Phe) mutant proteins, is already quenched in viscous low-temperature solutions, before the onset of major structural fluctuations in the macromolecule, an anomalous quenching that is abolished with the mutation Y283V (Tyr283-->Val). In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 micros to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction with Y283. Tyr quenching of W310 exhibits a strong temperature dependence, with a rate constant kq = 0.1 s(-1) at 140 K and 2 x 10(4) s(-1) at 293 K. Comparison between thermal quenching profiles of the W84F mutant in solution and in the dry state, where protein flexibility is drastically reduced, shows that the activation energy of the quenching reaction is rather small, Ea < or = 0.17 kcal mol(-1), and that, on the contrary, structural fluctuations play an important role on the effectiveness of Tyr quenching. Various putative quenching mechanisms are examined, and the conclusion, based on the present results as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an excited-state triplet exciplex.

Full Text

The Full Text of this article is available as a PDF (93.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bent D. V., Hayon E. Excited state chemistry of aromatic amino acids and related peptides. III. Tryptophan. J Am Chem Soc. 1975 May 14;97(10):2612–2619. doi: 10.1021/ja00843a004. [DOI] [PubMed] [Google Scholar]
  2. Cioni P., Strambini G. B. Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence. Signal transmission following binding of allosteric effectors. J Mol Biol. 1989 May 5;207(1):237–247. doi: 10.1016/0022-2836(89)90453-1. [DOI] [PubMed] [Google Scholar]
  3. Cioni P., Strambini G. B. Pressure effects on protein flexibility monomeric proteins. J Mol Biol. 1994 Sep 23;242(3):291–301. doi: 10.1006/jmbi.1994.1579. [DOI] [PubMed] [Google Scholar]
  4. Cioni P., Strambini G. B. Pressure effects on the structure of oligomeric proteins prior to subunit dissociation. J Mol Biol. 1996 Nov 15;263(5):789–799. doi: 10.1006/jmbi.1996.0616. [DOI] [PubMed] [Google Scholar]
  5. Gabellieri E., Rahuel-Clermont S., Branlant G., Strambini G. B. Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. Biochemistry. 1996 Sep 24;35(38):12549–12559. doi: 10.1021/bi960231b. [DOI] [PubMed] [Google Scholar]
  6. Gabellieri E., Strambini G. B. Conformational changes in proteins induced by dynamic associations. A tryptophan phosphorescence study. Eur J Biochem. 1994 Apr 1;221(1):77–85. doi: 10.1111/j.1432-1033.1994.tb18716.x. [DOI] [PubMed] [Google Scholar]
  7. Gonnelli M., Strambini G. B. Glycerol effects on protein flexibility: a tryptophan phosphorescence study. Biophys J. 1993 Jul;65(1):131–137. doi: 10.1016/S0006-3495(93)81069-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gonnelli M., Strambini G. B. Phosphorescence lifetime of tryptophan in proteins. Biochemistry. 1995 Oct 24;34(42):13847–13857. doi: 10.1021/bi00042a017. [DOI] [PubMed] [Google Scholar]
  9. Hansen J. E., Steel D. G., Gafni A. Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence. Biophys J. 1996 Oct;71(4):2138–2143. doi: 10.1016/S0006-3495(96)79414-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henis Y. I., Levitzki A. The role of the nicotinamide and adenine subsites in the negative co-operativity of coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase. J Mol Biol. 1977 Dec 15;117(3):699–716. doi: 10.1016/0022-2836(77)90065-1. [DOI] [PubMed] [Google Scholar]
  11. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  12. Li Z., Bruce A., Galley W. C. Temperature dependence of the disulfide perturbation to the triplet state of tryptophan. Biophys J. 1992 May;61(5):1364–1371. doi: 10.1016/S0006-3495(92)81943-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schauerte J. A., Steel D. G., Gafni A. Time-resolved room temperature tryptophan phosphorescence in proteins. Methods Enzymol. 1997;278:49–71. doi: 10.1016/s0076-6879(97)78006-6. [DOI] [PubMed] [Google Scholar]
  14. Skarzyński T., Wonacott A. J. Coenzyme-induced conformational changes in glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. J Mol Biol. 1988 Oct 20;203(4):1097–1118. doi: 10.1016/0022-2836(88)90130-1. [DOI] [PubMed] [Google Scholar]
  15. Smith C. A., Maki A. H. Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme. Biophys J. 1993 Jun;64(6):1885–1895. doi: 10.1016/S0006-3495(93)81560-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Strambini G. B., Gabellieri E. Intrinsic phosphorescence from proteins in the solid state. Photochem Photobiol. 1984 Jun;39(6):725–729. [PubMed] [Google Scholar]
  17. Strambini G. B., Gabellieri E. Phosphorescence anisotropy of liver alcohol dehydrogenase in the crystalline state. Apparent glasslike rigidity of the coenzyme-binding domain. Biochemistry. 1987 Oct 6;26(20):6527–6530. doi: 10.1021/bi00394a036. [DOI] [PubMed] [Google Scholar]
  18. Strambini G. B., Gabellieri E. Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys J. 1996 Feb;70(2):971–976. doi: 10.1016/S0006-3495(96)79640-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Strambini G. B., Gabellieri E. Temperature dependence of tryptophan phosphorescence in proteins. Photochem Photobiol. 1990 Jun;51(6):643–648. [PubMed] [Google Scholar]
  20. Strambini G. B., Gonnelli M. Effects of urea and guanidine hydrochloride on the activity and dynamical structure of equine liver alcohol dehydrogenase. Biochemistry. 1986 May 6;25(9):2471–2476. doi: 10.1021/bi00357a027. [DOI] [PubMed] [Google Scholar]
  21. Strambini G. B., Gonnelli M. Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution. Biochemistry. 1990 Jan 9;29(1):196–203. doi: 10.1021/bi00453a027. [DOI] [PubMed] [Google Scholar]
  22. Sun L., Kantrowitz E. R., Galley W. C. Room temperature phosphorescence study of phosphate binding in Escherichia coli alkaline phosphatase. Eur J Biochem. 1997 Apr 1;245(1):32–39. doi: 10.1111/j.1432-1033.1997.00032.x. [DOI] [PubMed] [Google Scholar]
  23. Vanderkooi J. M., Englander S. W., Papp S., Wright W. W., Owen C. S. Long-range electron exchange measured in proteins by quenching of tryptophan phosphorescence. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5099–5103. doi: 10.1073/pnas.87.13.5099. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES