XAS STUDIES OF METAL TRANSFER

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。 检测和定量分子伴侣与靶蛋白之间的金属转移反应是目前研究的热点。一个重要的实验要求是能够标记该对中的一个成员，以便在转移进行时跟踪结合金属的位置。在这一建议中，我们开发了一种策略，其中硒蛋氨酸(SEM)或硒半胱氨酸(SEC)取代伴侣蛋白靶对中一个成员的结合位点的天然Met或Cys配体。然后，通过在Se和Cu吸收边的X射线吸收光谱监测Se-Cu相互作用，Se标记可以用作转移过程中铜原子位置的直接光谱探针。我们的研究将包括(I)周质外排蛋白CusF和CusB之间的铜转移反应，(Ii)嗜热T细胞色素氧化酶CUA和Sco或PCuAC之间的铜转移反应，以及(Iii)哺乳动物CTR1导入蛋白和HCCS之间的铜转移反应。这项工作的目标是获得选择性转移化学的证据，测量过程的动力学，并使用这些数据来询问转移机制的基本结构元素。

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. Detecting and quantifying metal transfer reactions between chaperones and target proteins is currently of great interest. An important experimental requirement is the ability to label one member of the pair in order to track the location of the bound metal as the transfer proceeds. In this proposal we develop a strategy in which selenomethionine (SeM) or selenocysteine (Sec) are substituted for the native Met or Cys ligands of the binding sites of one member of the chaperone target pair. The Se label can then be used as a direct spectroscopic probe of the location of a copper atom during the transfer process via monitoring of the Se-Cu interaction by x-ray absorption spectroscopy at both the Se and Cu absorption edges. Our studies will include copper transfer reactions between (i) the periplasmic efflux proteins CusF and CusB, (ii) T thermophilus cytochrome oxidase CuA and Sco or PCuAC, and (iii) the mammalian CTR1 importer and hCCS. The goal of the work is to obtain evidence for selective transfer chemistry, measure the kinetics of the process, and use this data to interrogate the essential structural elements of the transfer mechanism.