Unfolding thermodynamics of the tetrameric chaperone, SecB

Abstract

SecB is a cytosolic tetrameric chaperone in Escherichia coli, which maintains polypeptides, destined for export in a translocation competent state. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. Increasing the pH decreases the stability of the tetramer significantly, the T_m changing from 341.3 K at pH 6.5 to 332.6 K at pH 9.5. The value of ΔC_p obtained from measurements of ΔH_m as a function of T_m was 10.7 ± 0.7 kcal mol⁻¹ K⁻¹. The value of ΔC_p is among the highest measured for a multimeric protein. At 298 K, pH 7.4, the ΔG°_u for the SecB tetramer is 27.9 ± 2 kcal mol⁻¹. Denaturant-mediated unfolding of SecB was found to be irreversible. The reactivity of the four solvent-exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well-folded, and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity.