STRUCTURAL INVESTIGATIONS OF METALLOPROTEIN METAL SITES

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DESCRIPTION: (Adapted from applicant's abstract) The long term objective of this proposal is to characterize in detail the structural and functional properties of Zn(II) in biological systems. Zinc is the most common metal found in metalloproteins and is the only metal that is known to be required for every major class of enzyme catalysis. Hundreds of zinc proteins have been isolated and thousands of potential zinc binding sites have been identified in protein sequences. Imbalances in the levels of Zn, or errors in its transport or regulation can have profound health consequence. Despite its importance, there is relatively little information available about biological Zn(II) sites due to the difficulty of studying this spectroscopically "silent" metal. X-ray absorption spectroscopy, one of the few methods able to provide structural information for non-crystalline materials, will be used to determine their structures. Three major, inter-related objectives are proposed: -X-ray absorption spectroscopy will be used to characterize the Zn binding sites in a series of important proteins, with particular emphasis on a novel class of Zn-alkyl transfer enzymes. -Detailed comparisons will be made of the metal binding sites in a series of structurally defined peptides. The objective of this second set of experiments is to determine the relative importance of metal stereochemical preference and protein structure in defining the structure of a metal binding site. For this work, structures will be compared for a series of spectroscopically silent d10 metal ions (Cu(I), (Ag(I), Cd(II), Hg(II)) and metalloids As(III). -X-ray absorption spectroscopy, x-ray microprobe imaging, microprobe spectroscopy, and x-ray microtomography will be used to characterize the role(s) of Zn in embryo development in zebra fish and Xenopus laevis. Spatially and temporally-resolved spectroscopy will be used to determine the Zn speciation in developing embryos. Coupled capillary-electrophoresis/x-ray fluorescence will be used to resolve and characterize Zn containing proteins.