El-Mashtoly Samir F, Yamauchi Seigo, Kumauchi Masato, Hamada Norio, Tokunaga Fumio, Unno Masashi
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
J Phys Chem B. 2005 Dec 15;109(49):23666-73. doi: 10.1021/jp054772z.
Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50-->Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.
光活性黄色蛋白（PYP）是一种细菌蓝光光感受器，暗态PYP（PYP(dark)）的光激发会触发一个涉及多个中间态的光循环。我们报告了在225 - 250 nm激发下PYP的紫外共振拉曼光谱，并研究了伴随假定的信号态PYP(M)形成的蛋白质结构变化。PYP(M) - PYP(dark)差异光谱显示了酪氨酸和色氨酸的几个特征，表明这些氨基酸残基的环境发生了变化。酪氨酸差异信号在Y8a和Y9a波段强度变化时显示出小的向上位移。虽然PYP中有五个酪氨酸残基，但根据不同激发波长下差异光谱的全局拟合分析和PYP(dark)的晶体结构，Tyr42和Tyr118被认为是差异信号的来源。对Thr50→Val突变体的进一步实验支持了Tyr42的环境变化。观察到的Y8a波段向上位移表明PYP(M)中Tyr42与发色团之间的氢键减弱或断裂。此外，从Y9a波段的向上位移推测Tyr42中OH基团发生了重新取向。对于色氨酸，在PYP(M)形成时，W3、W16和W18模式的拉曼谱带强度减弱。强度的损失归因于PYP(M)中色氨酸的暴露。PYP仅含有一个色氨酸（Trp119），其位于距活性位点超过10 Å处。因此，观察到的变化表明在从PYP(dark)转变为PYP(M)的过程中蛋白质发生了全局构象变化。这些结果与目前提出的PYP光循环机制一致。
