Structure-based functional annotation of microbial genomes

Three-dimenstional structure of bacterial proteins can frequently be predicted by structure prediction algorithms. However, the accuracy of the predictions requires confirmation with experimental structure determination. Further, there are still many bacterial proteins for which the structure that cannot be predicted by algorithms, particularly those found on genomic islands that are associated with pathogenic bacteria. This project will combine the structure prediction efforts of the Freddolino group at University of Michigan with experimental structure determination at Northwestern University in the research group of Karla Satchell. The Michigan group will identify proteins of unknown structure and provide a list of proteins of significant interest to the Satchell group. These proteins or domains of larger proteins will either be identified for confirmation of a structure prediction or proteins or domains of larger proteins for which a structure could not be predicted. The Satchell group will examine the structure prediction and amino acid sequence and then clone the gene or gene fragment. Cloning will be done using synthetic DNA cloning services. The overexpression plasmids will be transformed into E. coli and tested for protein expression. If a structure prediction exists, the protein will be purified following expression in Terrific Broth or similar for purification of native protein. If no prediction was possible, the protein will be expresed in minimal media supplemented with selenomethionine. Proteins that are difficult to express or are not soluble will be tested further with alternate expression conditions (such as media, temperature, and induction conditions) and with solubility agents. If necessary, a second clone will be ordered and expression and solubility reattempted. Soluble recombinant proteins will be purified and set up for crystallization trials. Crystallization will monitored for up to one year. When crystals form, crystals will be frozen in cryoprotectant and x-ray diffraction data collected. If the crystals diffract at 3.0 angstrom or below, the structure will be refined and the finished structure provided to the Freddolino group. Upon completion of the project or in advance of publication, structures will be deposited to the RCSB Protein Data Bank and diffraction data will be deposited to proteindiffraction.org or similar database. It is expected that 2-3 expression/purification/crystallization attempts will made per month for 24-36 proteins per year with an expected average of 30 proteins per year. The current success rate of the group is ~10% so it is expected these efforts will result in 2-3 structures determine per year for 10-15 structures determined in this 5 year project.