XAS STUDIES OF METAL TRANSFER

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 检测和定量分子伴侣和目标蛋白之间的金属转移反应目前引起了人们的极大兴趣。一个重要的实验要求是能够标记该对中的一个成员，以便在转移过程中跟踪结合金属的位置。在本提案中，我们开发了一种策略，其中硒代蛋氨酸 (SeM) 或硒代半胱氨酸 (Sec) 取代伴侣靶点对一个成员的结合位点的天然 Met 或 Cys 配体。然后，Se 标记可用作转移过程中铜原子位置的直接光谱探针，通过 Se 和 Cu 吸收边的 X 射线吸收光谱监测 Se-Cu 相互作用。我们的研究将包括 (i) 周质外排蛋白 CusF 和 CusB、(ii) 嗜热菌细胞色素氧化酶 CuA 和 Sco 或 PCuAC、以及 (iii) 哺乳动物 CTR1 输入蛋白和 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，测量该过程的动力学，并使用这些数据来询问转移机制的基本结构要素。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.