Ames J B, Ros M, Raap J, Lugtenburg J, Mathies R A
Department of Chemistry, University of California, Berkeley 94720.
Biochemistry. 1992 Jun 16;31(23):5328-34. doi: 10.1021/bi00138a012.
Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.
细菌视紫红质的时间分辨紫外共振拉曼光谱用于研究纳秒和毫秒时间尺度上的蛋白质结构变化。使用240nm的激发光来选择性增强酪氨酸的振动散射，以便能够检测其氢键和质子化状态的变化。纳秒和毫秒紫外拉曼差光谱均表明，在BR----K和BR----M转变过程中，没有酪氨酸残基改变其电离状态。然而，在BR----M紫外拉曼差光谱中，在1172和1615cm-1处观察到强度变化。在酪氨酸-d4标记的BR中，1615cm-1处的特征峰向下移动了25cm-1，这与其作为酪氨酸振动的归属一致。BR----M紫外拉曼差光谱中的强度变化很可能反映了当一个或多个酪氨酸残基与M412中的氢键受体相互作用更强时共振增强的增加。BR----M紫外拉曼差光谱中v7a特征峰（1172cm-1）的频率支持这一解释。视网膜结合口袋中Tyr-185和Asp-212的接近表明，M412中席夫碱的去质子化导致Tyr-185通过形成更强的氢键来稳定离子化的Asp-212。
