This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
The goal of this research is to determine the mechanism of polypeptide translocation catalyzed by the hexameric E. coli ClpA motor protein. Motor proteins are essential enzymes for a variety of cellular processes that require mechanical work. Therefore, an understanding of the mechanism of how such enzymes couple the energy from ATP hydrolysis to movement along a linear track is essential to our basic understanding of cell function. Of fundamental interest in examining how such motor proteins operate is determining how far the motor travels on its track per step (step-size), how much energy is used per step (ATP coupling efficiency), how fast does it take this step (overall rate and microscopic rate-constants), and what is the probability that the motor will remain on its track vs. dissociate (processivity). Despite the fundamental importance of these proteins in the life cycle of a cell, these parameters that describe the mechanism of polypeptide translocation are not known for a protein unfoldase such as ClpA. Determination of the mechanism of protein translocation catalyzed by the E. coli ClpA protein unfoldase will be accomplished by employing rapid mixing kinetic approaches. Using these approaches, this research will yield measurements of the step-size, coupling efficiency, processivity, and overall rates for protein translocation. To aid in the interpretation of these parameters the ClpA self association and nucleotide driven association process will be examined using thermodynamic and hydrodynamic techniques.
Broader Impacts This research will advance knowledge across different fields by enabling others to begin examining a variety of polypeptide translocases involved in such important cellular processes as ATP dependent proteolysis, refolding of protein aggregates, and protein translocation across membranes. This research presents opportunities for discovery by offering a vehicle for undergraduate, graduate, and postdoctoral research assistants from diverse backgrounds to apply an array of biophysical approaches and molecular biology techniques. This training occurs in the research lab and has translated into the classroom. The principal investigator is strongly committed to broadening the participation of underrepresented groups in science and the broader research community. This is being accomplished by actively seeking minority graduate students at universities and colleges across Alabama and Mississippi, including Historically Black Colleges and Universities. Additionally, the principal investigator is a member of the Society for the Advancement of Chicanos and Native Americans in Science (SACNAS). As such, the principal investigator and members of the research group will be attending the SACNAS annual national meeting in an attempt to recruit undergraduate, graduate and postdoctoral candidates from underrepresented groups. This research will enhance the infrastructure by maintaining established collaborations both within the Department of Chemistry as well as across University of Alabama at Birmingham campus. An additional benefit of this project to society is that it has the potential to yield individuals highly trained in the application of an array of biophysical and molecular biology techniques. This has the potential to globally impact science and technology because students trained in these techniques will be well sought after by both academia and industry worldwide.
