STRUCTURAL AND FUNCTIONAL GENOMICS OF PROTEIN FAMILIES

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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. The data collection activities carried out within the PXRR in this project are focused on medium-throughput structure determination and characterization, primarily of enzymatic mechanisms of proteins from prokaryotic and eukaryotic sources that are involved in a variety of biological processes. The specific goals of these projects are to (a) determine the structure of the apo-protein (b) determine the co-crystal structure with a variety of possible substrate/product/inhibitor or cofactor molecules to clarify the enzymatic mechanism and (c) perform additional biophysical and biochemical characterization and site-directed mutagenesis of these proteins in order to better understand their function and to complement the crystal structure. Bacterial proteins of both known and unknown function are predominantly selected from the three sequenced genomes of Escherichia coli. Some of the proteins are unique to both E. coli pathogenic strains, as well as to other pathogenic bacteria, and may represent potential targets for therapeutic intervention. Over 30 crystal structures of E. coli proteins have arisen from this these studies, and represent a broad range of metabolic processes. In addition to E. coli proteins, we are interested in bacterial enzymes from Helicobacter pylori and Campylobacter jejuni involved in pseudaminic acid other carbohydrates synthesis and the general N-glycosylation machinery. Finally, we have a long standing interest in glycosaminoglycan lyases from Flavobacterium heparinum, Bacteroides thetaiotaomicron and other species Most of our published results are based on structure of a native enzyme together with several complexes. Eukaryotic proteins are selected based on proteomics analysis of cellular organelles carried within the Montreal Proteomics Network (e.g. CREG, ENTH domain of enthoprotin) and based on comparative microarray data for normal and cancer cell lines carried within a large-scale NRCC project. Targets of interest were selected following TGF-b treatment of a cell line used as a model for metastasis. Genes that are signifcantly up- or down-regulated were selected for structural studies. In addition, we are interested in scaffolding proteins in signaling pathways and their complexes with binding partners (e.g. MP1-p14 complex).