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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05453212/
• 关键词: Resonance Raman; Time-resolvedd resonance Raman; Myoglobin; Cytochrome c oxidase; Molecular dynamics; タンパク質ダイナミクス

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

在酶促反应中，蛋白质构象的快速变化以及活性位点的催化反应都起着重要的作用，因为底物的掺入和产物的释放是酶反复发挥作用所不可缺少的。它们通过蛋白质的侧链或主链的快速重排来实现。本研究的目的是揭示蛋白质构象的快速变化伴随着蛋白质的功能，并在实践中，我们研究了细胞色素c氧化酶和肌红蛋白的反应中间体的共振拉曼光谱。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转运。该反应在CO与血红素铁结合后被抑制。在O_2饱和的缓冲溶液中，由CO结合的酶光解CO来引发酶促反应，在引发后的时间Δ t处观察到瞬态的拉曼光谱。 ...更多信息 关于反应。根据氧同位素敏感谱带出现的顺序，确定了反应过程为：Fe^<III>-O_2*Fe^<III>-O-OCu^<II><tautomer>Fe^V=O*Fe^ =O <IV>*Fe^<III>-OH，从而在细胞色素c氧化酶60年的研究史上取得了突破性进展。根据他们的说法，没有CO从蛋白质外部迁移到血红素铁中的途径。因此，蛋白质结构的快速重排应该伴随着配体结合。为了揭示蛋白质的瞬态结构，我们进行了泵浦/探测时间分辨共振拉曼实验的光解CO-肌红蛋白及其远端组氨酸突变体的重组。研究发现，Fe-CO伸缩模式（nu_<Fe-CO>）在490 cm^左右<-1>的物种比nu_在510 cm^左右的物种恢复得更快<Fe-CO><-1>。然而，490 cm ↑ <-1>[3]物质并不是作为510 cm ↑ [3]物质的前体产生<-1>的。为了了解蛋白质动力学及其与nu_频率的关系<Fe-CO>，我们进行了从头算分子轨道计算和分子动力学。少

项目成果

P.Jewsbury, S.Yamamoto, T.Minato, M.Saito, and T.Kitagawa: "The proximal residue largely determines the CO orientation in carbonmonoxy globin proteins. An ab initio study of a haem prosthetic unit." J,Am.Chem.Soc.116. 11586-11587 (1994)

P.Jewsbury、S.Yamamoto、T.Minato、M.Saito 和 T.Kitakawa：“近端残基在很大程度上决定了碳单氧球蛋白中的 CO 方向。血红素假体单元的从头开始研究。”

Y.Sakan,T.Ogur,T.Kitagawa,F.A.Fraunifelter,R.Mattera,M.Ikeda: "Time‐Resdved Resonance Raman Study on the Binding of Carbon Monokede to Recombinent Human Myoglobin and its Distal Histidine Mutants" Biochemistry. 32. 5815-5824 (1993)

Y. Sakan、T. Ogur、T. Kitakawa、F. A. Fraunifelter、R. Mattera、M. Ikeda：“碳单核与重组人肌红蛋白及其远端组氨酸突变体结合的时间共振拉曼研究”生物化学 32。 5815-5824 (1993)

S.Hirota, T.Ogura, K.Shinzawa-Itoh, S.Yoshikawa, M.Nagai, and T.Kitagawa: "Vibrational assignments of the FeCO unit of CO-bound heme proteins revisited : Observation of a new CO-isotope-sensitive Raman band assignable to the FeCO bending fundamental." J.P

S.Hirota、T.Ogura、K.Shinzawa-Itoh、S.Yoshikawa、M.Nagai 和 T.Kitakawa：“重新审视 CO 结合血红素蛋白的 FeCO 单元的振动分配：观察新的 CO 同位素-

T.Lian, B.Locke, T.Kitagawa, M.Nagai, and R.M.Hochstrasser: "Determination of Fe-CO geometry in the subunits of carbonmonoxy hemoglobin M Boston using femtosecond infrared spectroscopy." Biochemi stry. 32. 5809-5914 (1993)

T.Lian、B.Locke、T.Kitakawa、M.Nagai 和 R.M.Hochstrasser：“使用飞秒红外光谱测定碳单氧血红蛋白 M Boston 亚基中的 Fe-CO 几何形状。”

P.Jewsbury,S.Yamamoto,T.Minato,M.Saito,and T.Kitagawa: "The Proximal Residue Largely Determines the CO Orientation in Carbonmonoxy Globin Proteins.An ab intio Study of a Haem Prosthetic Unit" Journal of the American Chemical Society. 116. 11586-11587 (199

P.Jewsbury、S.Yamamoto、T.Minato、M.Saito 和 T.Kitakawa：“近端残留物很大程度上决定了碳单氧球蛋白中的 CO 方向。血红素假体单元的从头算研究”美国化学杂志