Future Direction
I shall continue our mechanistic characterization of E. coli Lon protease using a combination of kinetics and biophysical techniques. My lab will collaborate with Carin Stemper, assistant professor at Northwestern University and Borries Demeler, assistant professor at The University of Health Science Center at San Antonio to determine the functional oligomeric state of E. coli and human Lon proteases in the absence and presence of nucleotide, peptides and DNA. My lab will elucidate the ATPase mechanism of Lon in the absence and presence of peptide substrate primarily by pre-steady state enzyme kinetic techniques. I have also designed an experiment to monitor the pre-steady state time courses of my proposed peptide translocation step. A proteolytically inactive E. coli Lon mutant (S679W) has been generated for this study. Within the next year, my lab will also begin the kinetic characterization of E. coli Lon degrading a peptide substrate containing two cleavage sites. These experiments will provide insights into how Lon degrades a polypeptide such as casein or l N that contains multiple cleavage sites. In addition to studying E. coli Lon, my lab has also identified unique peptides that are substrates for human Lon. We shall utilize these substrates to study the kinetic mechanism of human Lon. In addition, the mechanism of how Lon selects for oxidized mitochondrial proteins such as aconitase will be investigated.
I shall also continue my collaboration with the Suzuki Lab in evaluating the functional role of human Lon. My lab will use a combination of synthetic and kinetic approaches to degenerate different kinds of mechanistic probes to study the mechanism of substrate selectivity by human Lon. Affinity labels will also be used to map the DNA binding site in the proease. In addition, my lab and the Suzuki lab will work closely together in using a proteomic approach to identify mitochondrial DNA binding proteins that interact with human Lon. We hope to construct some of the in vivo reaction pathways of Lon by identifying accessory proteins that interact with Lon in maintaining nucleic acid metabolism.
The collaboration with the Berdis Lab will continue to focus on synthesizing small libraries of non-natural nucleotide triphosphates to inhibit DNA synthesis. In addition, we shall perform structure-function relation study on T4 DNA polymerase to obtain insights into the mechanism by which polymerase incorporate non-natural nucleotides. Information obtained from these studies will serve as guide for designing more potent inhibitors. Furthermore, we shall expand our inhibition studies to other DNA polymerases such as the Klenow fragment and HIV reverse transcriptase, which may employ a slightly different mechanism in incorporating non-natural nucleotides across a DNA template.
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