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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子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与功能关系具有重要意义，因此一直是生物学中的一个重要课题。尽管使用各种光谱技术进行了深入的研究，但由于光谱技术的结构灵敏度的限制，涉及构象变化的中间结构的完整表征仍然难以实现。为了实现这一目标，我们提出了研究锌取代的细胞色素c（锌细胞色素c）和野生型细胞色素c（铁细胞色素c）的构象变化，通过使用时间分辨广角X射线散射（TR-WAXS）。 Cyt-c是一个小的单域血红素蛋白，具有104个氨基酸，并已被认为是一个有用的模型系统，用于研究结构动力学。在锌取代的细胞色素c的情况下，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Zn Cyt-c被光激发，则与锌配位的残基解离，伴随着蛋白质结构和Zn氧化态的变化。以前的一项研究，使用时间分辨荧光光谱表明，在光诱导配体解离后，在溶液中的锌细胞色素c的结构重组。(2)结果表明，轴向配体的解离从Zn（II）原子的激光照射诱导重组的蛋白质从其天然的折叠状态。在同一项研究中，确定了两个动力学组件：一个快速组件约120 ps的时间常数和一个缓慢的7 ns的时间常数。这些常数与五配位和四配位Zn Cyt-c中间结构的存在有关。(2)在这个拟议的实验中，我们计划使用TR-WAXS在pH 7和磷酸盐缓冲液条件下研究Zn Cyt-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，通过在与Zn Cyt-c相同的时间范围内比较测量野生型Fe Cyt-c，我们的目的是找出血红素基团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.