HIGH THROUGHPUT CHARACTERIZATION OF THE YEAST METALLOME

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DESCRIPTION (provided by applicant): Metal ions are known to play a variety of critical roles in biological systems, providing structural, catalytic, and signaling functions, and approximately one-third of all proteins are believed to be metalloproteins. However, there has never been a comprehensive survey of the distribution and function of metal ions in an organism, that is, of the complete "metallome". The overall goal of the project is to characterize the role of metal ions in biology, using Saccharomyces cerevisiae as an initial test organism. The first step in this project will be to develop methods for metallome screening by developing methodology for high throughput expression and purification of all of the fusion proteins in the glutathione-S-transferase - ORF (open reading frame) library from S. cerevisiae. The metal: protein ratios for these fusion proteins will be determined using a combination of ICP-MS for high-sensitivity metal analysis and x-ray fluorescence as the primary tool for high-throughput non-destructive metal analysis and these tools will be used to define the S. cerevisiae metallome. For selected metalloproteins, the local environment of the metal ion (oxidation state and ligation) will be determined using x-ray absorption spectroscopy. The methodology developed for the initial screening will then be used to investigate, on a metallome-wide scale, the consequences of variations in growth conditions. Two model systems will be used: growth under conditions of limited Zn as a model for the effect of metal ion concentration on metal distribution in the metallome; and growth in the presence of Pb(ll) as a model for the effect of non-natural metal ions on the metallome.