The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.
Biophys Chem
; 184: 29-36, 2013 Dec 31.
Article
en En
| MEDLINE
| ID: mdl-24036048
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.
Palabras clave
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
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Rec A Recombinasas
/
Temperatura
/
Escherichia coli
/
Desplegamiento Proteico
Idioma:
En
Revista:
Biophys Chem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos