RUI: Investigating Salt Induced Effects on RecA Unfolding and Nucleotide Binding

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The objective of this project is to identify protein structures that are involved in nucleotide binding and ion protein interactions that result in the high salt induced changes in RecA activity and stability. The Escherichia coli RecA protein is a DNA-dependent ATPase that requires formation of a nucleoprotein filament to perform the DNA strand-exchange reaction in DNA repair and homologous genetic recombination. Salt induced effects that modulate enzyme structure and function involve specific changes in protein structures that result in modified activity and protein-protein interactions. In order to investigate the origin of salt induced effects on RecA the PI will use difference infrared spectroscopy, circular dichroism and fluorescence to study how salt influences the aggregation and unfolding of RecA while simultaneously studying how salt modulates nucleotide binding and ATP hydrolysis. Difference infrared spectroscopy is a powerful yet underutilized technique that has the ability to elucidate changes induced by nucleotide-binding under a variety of solution conditions at the single amino acid level. Combining difference infrared and circular dichroism studies will enable undergraduate students to obtain complementary information about salt induced effects on stability and nucleotide binding. Despite decades of study on many seemingly well-characterized proteins, identifying the specific amino acids and protein structures that are involved in substrate binding, allosteric regulation and ion-mediated processes has continued to be elusive.All of the work in this project will be performed by undergraduates at James Madison University. In addition, the PI has an established history of working with deaf and hard-of-hearing students who will be recruited for research efforts. This project will be important in helping all the involved students gain valuable research experience and will aid in recruiting and retaining future scientists. Furthermore, the results of these studies will lead to a better understanding of not only RecA, but also the wide variety of proteins whose activity, stability and structure are modulated by the presence of salt.