PHOTOCHEMICAL ASSAYS OF CUPREDOXIN THERMODYMANICS--NOVEL METAL ION SENSORS

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Life depends on the ability of metal ions to augment the chemistry of proteins. Polypeptide and metals interact synergistically to engender essential biomolecular structures, modulated molecular stabilities sand redox-, acid/base-, and photochemistry otherwise unattainable with the limited properties or organic compounds. To evaluate the determinants of metalloprotein metal-ion specificity and their relationships to protein conformational stability and redox properties we propose to design a two-site recognition/reporter bioinorganic conformational stability and redox properties we propose to design a two-site recognition reporter bioinorganic system based on well-characterized cupredoxin templates, which will allow spectroscopic evaluation of metal binding and protein conformation. Energy/electron transfer between designed Ru(II)- and Re(I)- photosensitizers attached to the protein surface and metal-ions bound in the protein active site will be monitored to determine affinity constants between a series of transition metal ions and blood cooper proteins Systemic perturbation of the metal ions, by substitution, and of azurin and amicyanin templates, by both rationally directed mutagenesis and selection from combinatorial mutant libraries, will allow in-depth examination of the relationships among metal-ion specificity, oxidation-state preference, and native fold stability. The insight gained from these studies will pave the way for the development of a library of hybrid inorganic/bioinorganic sensors that can selective react with exogenous transition metals and in doing so produce unique optical signals diagnostic of exogenous metal type, oxidation state, and concentration.