The Escherichia coli protein RecA catalyzes DNA strand exchange and serves to facilitate DNA repair and genetic recombination. Nucleotide binding influences RecA oligomerization and its affinity for DNA. Past studies in our lab indicate dramatic buffer-specific changes in RecA stability and unfolding transitions. Circular dichroism (CD), infrared (IR), and fluorescence spectra show only minimal buffer-specific changes in nucleotide binding and secondary structure that do not account for the large differences in RecA stability and unfolding profiles. These observations led to further investigations of how the four common biological buffers Tris, HEPES, MES, and phosphate alter RecA structure and nucleotide binding. Here we have employed difference infrared spectroscopy utilized in conjunction with caged nucleotides to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. This higher resolution technique is aimed at detecting if the buffers alter nucleotide binding and also serves as an indirect probe to see if the buffers may promote RecA to higher order oligomerization states. Preliminary results show that ADP binding results in perturbations in Gln, Glu, Asp, Asn, Tyr, and Lys residues and secondary structural changes. Initial comparisons of difference spectra obtained in the four buffers show some similar changes but also show some differences. These variations between RecA-ADP minus RecA difference spectra will be discussed. Ultimately, this research is beneficial to RecA’s various applications in nanoscience, antibiotic resistance, and model protein systems.

Additional Abstract Information

Student(s): Joshua Temple

Department: Chemistry and Biochemistry

Faculty Advisor: Dr. Gina MacDonald

Type: Oral

Year: 2014

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