title: The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.
abstract: The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions.
authors.affiliation: Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA 22807, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Metrick
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Temple
authors.forename: Gina
authors.initials: G
authors.lastname: MacDonald
mesh_terms: D001665:Binding Sites; D002021:Buffers; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: D002021:Buffers; D004247:DNA; D011938:Rec A Recombinases
keywords: 2-(4-(2-hydroxyethyl)piperazin-1-yl) ethanesulfonic acid; 2-(N-morpholino) ethanesulfonic acid; 2′-3′-O-(N-methylanthraniloyl); ATPγS; Buffer effect; CD; Circular dichroism; DNA binding; EtBr; FRET; HEPES; MANT; MES; RecA; Thermal unfolding; Tris; adenosine 5′-O-(thiotriphosphate); circular dichroism; double-stranded DNA; dsDNA; ethidium bromide; fluorescence resonance energy transfer; pH effect; single-stranded DNA; ssDNA; tris(hydroxymethyl)aminoethane