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.
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