This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Sequencing the human and over fifty other genomes is only the first step in understanding the complex biological relationships that the raw genetic code forms the foundation of. Even in the yeast Saccharomyces cerevisiae, an extensively studied model organism, only 2/3 of the 5800 genes have proposed functions , and many of these are proposals based solely on homology modeling and not on characterization of purified proteins. To fill this gap, it is crucial to develop high-throughput methods to characterize proteins with respect to function and important biochemical properties including metal ion content and dependence. We used X-ray fluorescence (XRF) to determine the metal-binding properties of a large set of the 5800 predicted S. cervisiae proteins and to structurally characterize select, newly-discovered metalloproteins by EXAFS and XANES. These techniques provide information about the metal loading, oxidation state, and chemical environment of the metal bound to a metalloprotein information that is often not clear even from a high-quality crystal structure and therefore provide an important compliment to such techniques as high-throughput protein crystallography.We have determined the metal content of over 1500-3000 yeast proteins by XRF, and quantify the protein by an ELISA assay. We have measured the samples in triplicate to optimize the statistical results and minimize experimental errors. The collected data is under analysis.
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