X-ray absorption spectroscopy (XAS) will be used, as a component of multidisciplinary biochemical and biophysical structure/function studies, to provide high-resolution local structural information about metal sites in a number of metallobiochemical systems. Proposed studies focus on the unique contribution to be made by XAS in studying """"""""spectroscopically difficult"""""""" metals, including Zn, Cd, Hg, As, Sb. Many of the proposed experiments involve the role of metals in the important biological processes of transcription and its metalloregulation, heavy metal toxicity and resistance, and metal homeostasis. In particular, presumptive metal-binding motifs in basal eucaryal and archaeal transcription factors will be examined for metal binding, specificity, and requirement. The initial target for this effort is TFIIB. Other experiments will characterize the metal-binding residues and coordination environments of the metalloregulatory proteins MerR, ArsR, ArsD, and CadC. The correlation of metal coordination changes and allosteric regulation associated with protein conformational changes will be studied. Site-directed mutagenesis designed to alter the specificity for metal inducers, creating a Cd(II)-sensitive ArsR, for example, will require XAS for metal site characterization. Biophysical characterization of metal-binding affinity and specificity of MerP, a prototypical example of a widely distributed ca. 40-residue sequence motif containing a GmtCxxC sequence (Met2 and Thr3 are nearly conserved) will be carried out. The occurrence of variants of this """"""""heavy metal associated"""""""" (HMA) motif in a family of CPx-type ATPases, all of which are involved in heavy metal efflux or homeostasis for mercury, cadmium, and copper (at least), makes a characterization of their specificity particularly attractive. Six copies of these HMA motifs occur near the N-termini of the products of the MNK and WND genes, which have been associated with the copper-based Menkes and Wilson's diseases, respectively. The use of directed evolution to develop the MerP HMA motif into a specific recognition domain for any given metal ion will require XAS to characterize the metal coordination.
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