PUMP-PROBE X-RAY SOLUTION SCATTERING TO REVEAL CONFORMATIONAL CHANGE OF ZINC

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 蛋白质结构动力学的研究对于理解蛋白质的结构-功能关系具有重要意义，一直是生物学中的重要课题。尽管使用各种光谱技术进行了密集的研究，但由于光谱技术的结构敏感性的限制，涉及构象变化的中间结构的完整表征仍然是难以捉摸的。为了实现这一目标，我们提出用时间分辨广角X射线散射技术研究锌取代细胞色素c和野生型细胞色素c的构象变化。 Cyt-c是一种含104个氨基酸的单结构域的小分子血红素蛋白，被认为是研究结构动力学的有用模型系统。对于锌取代的细胞色素c，其血红素基团中的锌原子通常同时与His 18残基和Met 80残基共价配位。(图1)(1)然而，如果锌Cyt-c被光激发，与锌配位的残基就会解离，伴随着蛋白质结构的变化和锌的氧化态。此前，一项利用时间分辨荧光光谱的研究表明，在光诱导配体解离后，溶液中的锌细胞色素c发生了结构重组。(2)实验结果表明，激光作用下，锌原子上的轴向配体发生解离，导致蛋白质从天然折叠状态发生重组。在同一项研究中，确定了两个动力学成分：一个约为120ps时间常数的快成分和一个约为7 ns时间常数的慢成分。这些常数与五配位和四配位的锌Cyt-c中间结构的存在有关。(2)在本实验中，我们计划在pH=7和磷酸盐缓冲条件下用TR-WAXS研究锌细胞色素C的构象动力学。这项研究将使我们能够直接跟踪与配体解离相关的蛋白质构象变化。此外，通过对野生型Fe Cyt-c与锌Cyt-c在同一时间范围内的对比测量，我们旨在找出血红素中金属离子的类型与构象动力学之间的关系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The study of protein structural dynamics has been an important subject in biology owing to its significance in understanding the structure-function relationship of the proteins. Despite intense investigations using various spectroscopic techniques complete characterization of intermediate structures involved in a conformational change still remains elusive due to the limitation in structural sensitivity of the spectroscopic techniques. To achieve that goal we propose to investigate the conformational change of zinc-substituted cytochrome c (Zn Cyt-c) and wild type cytochrome c (Fe Cyt-c) by using time-resolved wide-angle X-ray scattering (TR-WAXS). Cyt-c is a small single-domain heme protein with 104 amino acids and has been considered a useful model system for studying structural dynamics. In case of zinc-substituted cytochrome c the Zn atom in its heme group is usually covalently coordinated with His 18 residue and Met 80 residue simultaneously. (Figure 1) (1) However if Zn Cyt-c is photoexcited the residue coordinated with zinc is dissociated accompanying the change of the protein structure and the Zn oxidation state. Previously a study using time-resolved fluorescence spectroscopy suggested the structural reorganization of Zn Cyt-c in solution after photoinduced ligand dissociation. (2) The results indicate that the dissociation of axial ligands from Zn(II) atom by laser irradiation induces the reorganization of the protein from its native folded state. In the same study two kinetic components were identified: a fast component of about 120 ps time constant and a slow one of 7 ns time constant. These constants are related with the existence of penta- and tetra-coordinated Zn Cyt-c intermediate structures. (2) In this proposed experiment we plan to investigate the conformational dynamics of Zn Cyt-c using TR-WAXS under pH7 and phosphate buffer condition. This study will allow us to directly follow the protein conformational changes associated with the ligand dissociation. In addition by comparatively measuring the wild type Fe Cyt-c in the same time range as for Zn Cyt-c we aim to find out the relationship between the type of a metal ion in the heme group and the conformational dynamics.