The long term objective of this proposal is to characterize in detail the structural and functional properties of Zn 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. Zinc appears to play an important role in a wide range of biological functions, ranging from embryonic development to active cell death. Despite this importance, there is relatively little information available about zinc sites due to the difficulty of studying spectroscopically """"""""silent"""""""" metals, such as Zn(II). X- ray absorption spectroscopy, one of the few methods able to provide structural information for non-crystalline materials, will be used to determine zinc site structures. Problems in assigning coordination spheres of zinc from XAS data are recognized and dealt with. Four major, inter-related objectives are proposed. First, extended X-ray absorption fine structure (EXAFS) will be used to characterize the Zn (and Hg and Cd) binding sites in a series of important proteins, including alanine tRNA synthetase, tRNA guanine transglycosylase, cobalamin-independent methionine synthase, mercuric reductase, and several Hg-specific antibodies. Second, detailed comparisons will be made of the Zn-, Cd- and Hg- 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. Third, methodology will be developed for measuring polarized x-ray absorption spectra of DNA-binding proteins. This approach will be based on using DNA as a scaffolding that can provide the protein orientation necessary for polarized spectroscopy. This methodology will be used to characterize the Zn, Cd and Hg sites in the metalloregulatory protein, MerR, in order to understand the origin of metal specificity in MerR. Fourth, x-ray absorption spectroscopy and micro x-ray fluorescence imaging will be used to characterize the role(s) of Zn in embryo development in zebra fish and Xenopus laevis. Temporal resolution will be used to define the changes in Zn structure that take place following oocyte fertilization. Spatial resolution (imaging) will be used to characterize the transport of Zn during development. Spatially- and temporally- resolved spectroscopy will be used to determine Zn speciation in developing embryos.
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