Elucidation of Dynamical Structures of Proteins by Ultraviolet Resonance Raman Spectroscopy

蛋白质的动力学结构对于理解蛋白质的结构-功能关系具有重要意义。由于振动光谱对分子几何结构敏感且时间响应足够快，我们采用紫外共振拉曼技术选择性地探测了200-240 nm左右的芳香残基的振动。首先，建立了实验系统。将Nd:YAG激光的四次谐波引入1 m的氢拉曼移位器中，分离出218 nm的辐射。拉曼散射是用一个太阳盲光电倍增管连接在一个1.26米的单轴上进行检测的。二级采用2400 gr/mm的光栅。在该系统中，通过观察芳香残基的共振增强拉曼带，研究了羰基血红蛋白（COHb）光解后的季元结构动力学特征。拉曼散射是兴奋10纳秒脉冲在218海里,从secon获得 ... d H_2喇曼转变的第四谐波的Nd: YAG激光器,而公司被10-ns photodissociated脉冲在419 nm由氮激光器泵浦染料激光器产生或产生的脉冲在436 nm第一H_2喇曼转变二次谐波的Nd: YAG激光光解脉冲的延迟时间(DELTAt_d)的拉曼探针脉冲是不同的从-100年到500亩。在DELTAt_d =-100 μ s和10 μ s时，观察到的光谱与不加泵浦光束时的光谱相同，这与先前报道的蛋白结构在7 μ s时变化的结果相反，但当DELTAt_d从10 μ s变化到20 μ s时，光谱模式确实发生了变化；1613和1011 cm^<-1>波段强度减弱，878 cm^<-1>波段强度转变为883 cm^<-1>。这些光谱变化表现为平滑单调的时间函数，表明在DELTA_d = 10 μ m附近不存在中间体。在加入一种效应物（六磷酸肌醇）后，也观察到类似的光谱变化。因此，观察到的紫外共振拉曼光谱的变化归因于四级结构的变化，可能是由于α 1- β 2亚基界面上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