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Detection of Fast Conformation Changes of Proteins by Time-Resolved Resonance Raman Spectroscopy

通过时间分辨共振拉曼光谱检测蛋白质的快速构象变化

基本信息

批准号：
05453212
负责人：
KITAGAWA Teizo
金额：
$ 4.8万
依托单位：
Institute for Molecular Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Fast conformation changes of proteins in addition to the catalytic reaction at the activie site play an important role in enzymic reactions, since the incorporation of substrates and release of products are indispensable for enzymes to function repeatedly. They are achieved by rapid rearrangements of side chains or main chain of proteins. It is the purpose of this study to reveal such fast conformation changes of protein accompanied by functioning of proteins, and in practice, we investigated resonance Raman spectra of reaction intermediates of cytochrome c oxidase and myoglobin.Cytochrome c oxidase is the terminal enzyme of mitochondrial respiration chain and catalyzes the reduction of molecular oxygen coupled with proton translocation. This reaction is inhibited upon binding of CO to the heme iron. The enzymic reaction was initiated by photolyzing CO from CO-bound enzymes in O_2-saturated buffer solution, and transient Raman spectra were observed at time DELTAt following the initiati … More on of reaction. On the basis of the order of appearance of oxygen-isotopesensitive bands, it has been established that the reaction proceeds in the following way ; Fe^<III>-O_2*Fe^<III>-O-OCu^<II><tautomer>Fe^V=O*Fe^<IV>=O*Fe^<III>-OH.Thus, this study has made a breakthrough in 60 years of research history of cytochrome c oxidase.The molecular structures of myoglobin has been revealed with x-ray crystallography at the level of 1.5 A resolution for both deoxy- and CO-bound forms. According to them, there is no pathway for migration of CO into the heme iron from outside of the protein. Therefore, rapid rearrangements of protein structures should be accompanied by the ligand binding. In order to reveal transient structures of proteins, we carried out the pump/probe time-resolved resonance Raman experiments for recombination of photodissociated CO-myoglobin and its distal histidine mutants. It was found that the species with the Fe-CO stretching mode (nu_<Fe-CO>) around 490 cm^<-1> recover much faster than those with nu_<Fe-CO> around 510 cm^<-1>. However, the 490 cm^<-1> species was not generated as a precursor of the 510 cm^<-1>species. To understand the protein dynamics and their relation with the nu_<Fe-CO> frequency, we carried out ab initio MO calculations and molecular dynamics. Less

蛋白质的快速构象变化和活性部位的催化反应在酶反应中起着重要的作用，因为底物的结合和产物的释放是酶重复发挥功能所不可缺少的。它们是通过蛋白质的侧链或主链的快速重排实现的。本研究的目的是揭示伴随着蛋白质功能的这种快速构象变化，并在实践中研究了细胞色素c氧化酶和肌红蛋白的反应中间产物的共振拉曼光谱。细胞色素c氧化酶是线粒体呼吸链的末端酶，催化分子氧的还原和质子的移位。当一氧化碳与血红素铁结合时，该反应被抑制。在O_2饱和的缓冲溶液中，通过光解CO结合的酶中的CO来引发酶反应，并在启动…之后的时间DELTAt观察到了瞬时拉曼光谱更多关于反应的信息。根据氧同位素敏感带的出现顺序，已确定该反应按以下方式进行：Fe^&lt；III&gt；-O_2*Fe^&lt；III&gt；-O-OCu^&lt；II&gt；&lt；tautomer&gt；Fe^V=O*Fe^&lt；IV&gt；=O*Fe^&lt；III&gt；因此，这项研究在细胞色素c氧化研究60年的历史上取得了突破，用1.5A分辨率的X射线结晶学揭示了肌红蛋白的脱氧和CO结合形式的分子结构。根据他们的说法，一氧化碳没有从蛋白质外部迁移到血红素铁的途径。因此，蛋白质结构的快速重排应该伴随着配体的结合。为了揭示蛋白质的瞬时结构，我们对光解CO-肌红蛋白及其远端组氨酸突变体的重组进行了泵浦/探测时间分辨共振拉曼实验。结果表明，在490 cm；-1&gt；附近具有Fe-CO伸展模式的物种比在510 cm；-1&gt；附近具有伸展模式的物种恢复得更快。然而，490 cm；-1&gt；种并不是510 cm；-1&gt；种的前身。为了了解蛋白质的动力学及其与Nu&lt；Fe-CO&gt；频率的关系，我们进行了从头算和分子动力学计算。较少