Elucidation of Dynamical Structures of Proteins by Ultraviolet Resonance Raman Spectroscopy

蛋白质的动态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的几何结构敏感，且响应时间足够快，我们采用紫外共振拉曼技术选择性地探测了芳香族残基的振动，其吸收带在200-240 nm附近。首先，确定了一个实验系统。将四次谐波引入1m氢拉曼移位器中，实现了对218 nm激光的隔离。用1.26米单色器上的太阳盲光倍增管测量拉曼散射。第二个顺序使用2400gr/mm的光栅。利用该系统，通过观察芳香族残基的共振增强拉曼光谱，研究了碳氧合血红蛋白(COHb)光解后四级结构的动力学特征。用秒…获得了波长为2 18 nm的10 ns激光脉冲，激发了拉曼散射当CO被氮气激光泵浦的染料激光器产生的419 nm处的10 ns脉冲或由其二次谐波的第一个H_2拉曼位移产生的436 nm的脉冲光解时，从光解脉冲到拉曼探测脉冲的延迟时间(DELTAt_D)在-100~500微秒之间变化。当DELTAt_d=-100mus和10 ns时，观察到的光谱彼此相同，与基于蛋白质结构7 ns变化的预期相反，但当DELTAt_d从10变化到20mus时，光谱模式发生了变化；1613和1011 cm^&lt；-1&gt；的谱带强度降低，878 cm^&lt；-1&gt；的谱带移动到883 cm^&lt；-1&gt；这些光谱变化表现为时间的光滑单调函数，表明在Delta_d=10mus附近没有中间体。在metHbF中加入效应剂(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元结构的变化，可能是由于α1-β2亚基界面上的β37-Trp和α42-Tyr的状态变化。较少

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