Strambini G B, Gabellieri E, Gonnelli M, Rahuel-Clermont S, Branlant G
Istituto di Biofisica, C.N.R., Pisa, Italy.
Biophys J. 1998 Jun;74(6):3165-72. doi: 10.1016/S0006-3495(98)78022-1.
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.
已知酪氨酸可猝灭溶液中游离色氨酸衍生物的磷光，但蛋白质中色氨酸残基与相邻酪氨酸侧链之间的相互作用尚未得到证实。本报告通过比较野生型酶与适当设计的突变体（其中第二个色氨酸残基W84被苯丙氨酸取代或Y283被缬氨酸取代）的磷光特性，研究了嗜热脂肪芽孢杆菌甘油醛-3-磷酸脱氢酶中Y283对W310磷光发射猝灭的潜在作用。多元醇/缓冲液低温玻璃中的磷光光谱和寿命表明，在野生型和W84F（Trp84→Phe）突变蛋白中，W310在粘性低温溶液中，在大分子主要结构波动开始之前就已经被猝灭，这种异常猝灭在Y283V（Tyr283→Val）突变时被消除。在室温缓冲液中，用缬氨酸取代Y283对W310磷光的影响是将其寿命从50微秒延长至2.5毫秒，增强了50倍，这再次强调了W310发射如何受与Y283的局部相互作用主导。W310的酪氨酸猝灭表现出强烈的温度依赖性，在140 K时速率常数kq = 0.1 s(-1)，在293 K时为2×10(4) s(-1)。溶液中和干燥状态下W84F突变体的热猝灭曲线比较表明，猝灭反应的活化能相当小，Ea≤0.17 kcal mol(-1)，相反，结构波动对酪氨酸猝灭的有效性起重要作用。研究了各种假定的猝灭机制，基于目前的结果以及其他蛋白质系统的磷光特性得出的结论是，酪氨酸猝灭是通过形成激发态三重态激基复合物发生的。
