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Strambini G. B., Gabellieri E., Gonnelli M., Rahuel-Clermont S., Branlant G. Tyrosine Quenching of Tryptophan Phosphorescence in Glyceraldehyde-3-Phosphate Dehydrogenase from Bacillus stearothermophilus. In: Biophysical Journal, vol. 74 (6) pp. 3165 - 3172. Biophysical Society, 1998.
 
 
Abstract
(English)
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 (Trp84right-arrowPhe) 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 (Tyr283right-arrowVal). In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 s 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 2 104 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 <= 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.
URL: http://www.biophysj.org/cgi/content/abstract/74/6/3165?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&author1=strambini&searchid=1&FIRSTINDEX=10&resourcetype=HWCIT
Subject Quenching
Tryptophan
Glyceraldehyde-3-phosphate dehidrogenase
Protein flexibility


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