Senior Honors Projects, 2010-2019
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Date of Graduation
Spring 2015
Document Type
Thesis
Degree Name
Bachelor of Science (BS)
Department
Department of Chemistry and Biochemistry
Advisor(s)
Gina MacDonald
Abstract
The Escherichia coli protein RecA catalyzes the strand exchange reaction used in DNA repair and genetic recombination. RecA is also a target for inhibiting microbial antibiotic resistance, understanding cancer propagation, and characterizing neurodegenerative disorders. Therefore, understanding factors that affect RecA structure and stability is broadly applicable to many fields. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. These studies suggest only minimal buffer-dependent changes in nucleotide binding and secondary structure but do not explain the significant differences in RecA stability and unfolding profiles. Here we have employed various biochemical and spectroscopic techniques to further characterize RecA both structurally and functionally in four common buffers: Tris, MES, HEPES, and Phosphate. Activity assays reveal RecA activity may be slightly decreased in HEPES and Phosphate buffers as compared to Tris and MES. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy show conservation of global RecA secondary structure in all buffers, yet unique aggregation states are observable in CD turbidity plots. Laser-induced photolysis of caged nucleotides was also used in conjunction with difference FTIR spectroscopy to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. These studies detected unique buffer-specific changes in nucleotide binding to RecA in each buffer and may provide insight into mechanisms for buffer-specific stability profiles of RecA.
Recommended Citation
Temple, Joshua E., "Probing buffer-specific effects on nucleotide binding to RecA using difference Fourier transform infrared spectroscopy" (2015). Senior Honors Projects, 2010-2019. 115.
https://commons.lib.jmu.edu/honors201019/115
