Elucidation of Dynamical Structures of Proteins by Ultraviolet Resonance Raman Spectroscopy

蛋白质的动力学结构是理解蛋白质结构-功能关系的基础。由于振动光谱对分子的几何结构很敏感，并且时间响应足够快，我们采用紫外共振拉曼技术选择性地探测芳香残基在200-240 nm附近的振动。首先，建立了一个实验系统。将Nd：YAG激光的四次谐波引入1 m氢拉曼移相器，分离出218 nm的辐射。拉曼散射检测与太阳盲光电倍增管连接到一个1.26米的单单色仪。在第二阶中使用具有2400 gr/mm的光栅。利用该系统，通过观察芳香残基的共振增强拉曼谱带，研究了一氧化碳血红蛋白（COHb）光解后四级结构的动力学特征。用10 ns的218 nm激光脉冲激发拉曼散射， ...更多信息 用氮激光泵浦染料激光器产生的419 nm的10 ns脉冲或Nd：YAG激光器的二次谐波的第一次H_2拉曼拉曼位移产生的436 nm的脉冲光解CO。从光解脉冲到拉曼探测脉冲的（DELTAt_d）在-100到500 μ s之间变化。对于DELTAt_d =-100 μ s和10 ns获得的观测光谱彼此相同，并且与没有泵浦光束时获得的光谱相同，这与基于蛋白质结构的7-ns变化的报道的预期相反，但是当DELTAt_d从10到20 μ s变化时，光谱图案确实改变; 1613和1011 cm ↑ [2]处的谱带<-1>强度降低，878 cm ↑ [2]处的谱带<-1>移动到883 cm ↑ [2]<-1>。这些光谱的变化出现作为一个平滑的单调函数的时间，这表明没有一个中间左右的DELTA_d = 10 μ s。一个类似的光谱变化也观察到后，除了效应（肌醇六磷酸）metHbF。因此，所观察到的UV共振拉曼光谱的变化归因于四级结构的变化，推测为α 1-β 2亚基界面处β 37-Trp和α 42-Tyr的状态变化。少

Dynamical structures of proteins is substantial to understand a structure-function relationship of proteins. Since vibrational spectroscopy is sensitive to a geometrical structure of molecules and its time response is sufficiently fast, we adopted ultraviolet resonance Raman technique to probe selectively the vibrations of aromatic residues with absorption bands around 200-240 nm. First, an experimental system was settled. The fourth harmonic of Nd:YAG laser was brought into a 1 m hydrogen Raman shifter, and the 218 nm radiation was isolated. Raman scattering was detected with a solar blind photomultiplier attached to a 1.26 m single monochomator. A grating with 2400 gr/mm was used in the second order. With this system dynamical features of quaternary structure of carbonmonoxy hemoglobin (COHb) after photolysis were pursued by observing resonance-enhanced Raman bands of the aromatic residues. The Raman scattering was excited by 10 ns pulses at 218 nm, which were obtained from the secon … More d H_2 Raman shift of the fourth harmonic of a Nd:YAG laser, while CO was photodissociated by 10-ns pulses at 419 nm generated by a nitrogen-laser pumped dye laser or by pulses at 436 nm generated from the first H_2 Raman shift of the second harmonic of the Nd:YAG laser The delay time (DELTAt_d) from the photolysis pulse to the Raman probe pulse was varied from -100 to 500 mus. The observed spectra obtained for DELTAt_d =-100 mus and 10 ns were the same as each other and as that obtained without the pump beam, contrary to expectations based on the reported 7-ns change of the protein structure, but the spectral pattern did change when DELTAt_d changed from 10 to 20 mus; the bands at 1613 and 1011 cm^<-1> decreased in intensity, and the band at 878 cm^<-1> shifted to 883 cm^<-1>. These spectral changes appeared as a smooth monotonous function of time, suggesting the absence of an intermediate around DELTA_d = 10 mus. A similar spectral change was also observed upon addition of an effector (inositol hexaphosphate) to metHbF. Accordingly, the observed changes of the UV resonance Raman spectra were attributed to a quaternary structure change, presumably to a status change of beta37-Trp and alpha42-Tyr at the alpha1-beta2 subunit interface. Less