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Scientific Statement
The goal of my research program is to study enzymes involved in stress-related protein
degradation and nucleic acid metabolism in order to find out ways to control cell growth.
To this end, we propose to develop a therapeutic cocktail that contains agents to inhibit
both protein degradation and nucleic acid metabolism. At present, we are focusing on
the enzymology of an ATP-dependent protease Lon, which functions to removedamaged proteins and certain short-lived regulatory nucleic acid binding proteins in the
cells. In addition, we are designing and synthesizing non-natural nucleotides to
inhibit damaged DNA synthesis mediated by low fidelity polymerases. My overall
strategy is to control the growth of an organism by simultaneously inhibit its protein
degradation and DNA synthesis pathway.
Research Contributions from my lab:
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Developed a fluorescent peptide substrate to study the kinetic mechanism of Escherichia coli (E. coli) Lon. This is the first synthetic peptide reported to stimulate the ATPase activity of Lon during its degradation. This peptide has also been used to characterize the enzymatic activities of mammalian Lon. |
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Constructed a testable kinetic model to account for the ATP-dependent peptide cleavage reaction of E. coli Lon using the steady-state and product inhibition data obtained in my lab. Our model hypothesizes that Lon couples ATP hydrolysis with the delivery of peptide substrate to the protease site prior to peptide cleavage (peptide translocation). |
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Demonstrated that E. coli Lon adopted a "closed conformation" when bound to adenine nucleotide; but this conformational change alone cannot activate the peptidase activity of Lon. Furthermore, the chemical structure of the nucleotide base dictates the affinity Lon for the nucleotide. |
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Demonstrated that nucleotide hydrolysis contributed to the catalytic turnover (k cat ) of peptide cleavage; and that ATP hydrolysis occurs before peptide cleavage during the first turnover of the ATP-dependent peptide or protein degradation. |
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Detected burst and lag kinetics in the pre-steady state time courses of ATP and peptide hydrolysis, respectively. The activation of peptidase activity in Lon is coordinated with the build up of an enzyme intermediate formed after ATP hydrolysis. |
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Generated non-natural nucleotides as probes to study the molecular mechanism of translesion DNA synthesis. |
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