| The focus of my research was functional characterization of three different protein systems by biochemical techniques and X-ray crystallography. The first project involved structural studies of the enzyme peptide deformylase (PDF) from Escherichia coli. What makes E. coli PDF different from other enzymes of the same class is its use of iron as opposed to zinc at its catalytic center. To elucidate the basis of this unique metal preference, we have solved high-resolution structures of PDF bound to three different metals---iron, cobalt, or zinc. Our structures reveal differences in how the product of the reaction---formate, binds to iron vs. zinc, and provide insight into PDF's mechanism of catalysis. These results have strong implications for gaining insights into the catalytic properties of PDF and subsequent development of antibiotics targeted against bacterial PDFs.; X-ray crystallography was also used to characterize proteins from a methane-generating microorganism called Methanosarcina barkeri. M. barkeri is an archaea that plays an essential role in the global carbon cycle by converting atmospheric carbon dioxide to methane. A previously unobserved amino acid, L-pyrrolysine was identified in the M. barkeri protein MtmB by our group and the laboratory of Dr. Joseph Krzycki (The Ohio State University, Microbiology). My focus in this collaborative project was to gather structural evidence for the proposed biological role of L-pyrrolysine in MtmB. As a part of this effort, we have determined the X-ray structure of the corrinoid protein MtmC that interacts with MtmB. Additionally, a model for the interaction between MtmB and MtmC was developed. This model is based on low-resolution X-ray data, homology modeling, and mass spectrometry.; The final system under investigation involves the study of putative protease networks in E. coli. Protein degradation is an important part of cellular metabolism in E. coli. Most of the initial steps of degradation are performed by ATP-dependent proteases: HflA, HflB (FtsH), Lon, and Clp (ClpXP, ClpAP, and ClpYQ or HslUV). The aim of this work is to explore a putative role of the peptidase oligopeptidase A as a downstream partner of these energy dependent proteases. Our results indicate that oligopeptidase A efficiently cleaves the peptides generated by the activity of three energy dependent proteases suggesting a potential role in multiple catabolic pathways. |
