Probing Buffer-Specific Effects on Nucleotide Binding to RecA using Difference Ftir

Abstract

The Escherichia coli protein RecA catalyzes the strand exchange reaction used in DNA repair and genetic recombination. Previous studies in our lab have shown buffer-specific changes in RecA stability and unfolding transitions. However, these studies suggest only minimal buffer dependent changes in nucleotide binding and secondary structure that did not explain the large buffer dependent differences in RecA stability and unfolding profiles. These observations led to further investigations of how the four common biological buffers Tris, HEPES, MES, and PO4 alter RecA structure and nucleotide binding. Here we have employed circular dichroism (CD) and infrared (IR) spectroscopy to further discern if buffers influence nucleotide binding to RecA. CD spectra of RecA were obtained in the presence and absence of ADP in each buffer condition. Laser-induced photolysis of caged nucleotides was used in conjunction with difference IR to generate RecA-ADP minus RecA difference infrared spectra in each of the four buffers. These studies detected buffer-specific changes in nucleotide binding to RecA including possible perturbations in Gln, Glu, Asp, Asn, Tyr, and Lys residues and unique secondary structural transitions. These differences between RecA-ADP minus RecA difference spectra will be discussed.