TCP1: ANALYSIS OF LYSINE MODIFICATION USING PROTEIN MICROARRAYS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Technology Core Projects 1 (TCP-1) Protein microarrays: Developing new protein microarray technology to identify protein protein interactions, small moleculeprotein interactions, enzyme substrates, specificity of binding and enzymatic reactions. A key new aspect of the technology to be developed here is to use in vitro translation systems as the starting point for protein production, which can significantly increase yields. We propose to investigate the optimal reaction conditions for analyzing lysine modifications, to identify downstream targets of different (de)acetylases, methylases, and (de)ubiquitylases in yeast, to create E. coil and human mitochondrial protein microarrays for analyzing specific protein acetylation activities, and to improve current protein microarray technologies. These studies are expected to provide a global picture of regulating protein functions through different kinds of posttranslational modifications on lysine side chains, and to further improve the application of protein microarray technologies. Specifically, we plan the following interrelated activities: 1) To fabricate yeast proteome microarrays and systematically test the optimal assay conditions for different kinds of bioassays related to lysine modification activities. 2) To construct an expression library containing all genes in the genome of E. coli, and purify their encoded proteins to create an E. coli proteome microarray; use these E. coli proteome microarrays to screen for substrates of the newly identified E. coil acetylase and deacetylase. 3) To construct a prioritized human mitochondrial protein chip to identify the interacting partners of human Sirt3p deacetylase and identify the corresponding acetyltransferase(s). 4) To further improve the protein microarray technologies as a faster, easier and userfriendlier approach that can be coupled to other detection met