Unfolding thermodynamics of the tetrameric chaperone, SecB

SecB is a cytosolic tetrameric chaperone in Escherichia coli, which maintai ns polypeptides, destined for export in a translocation competent stale. Th e 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 reve rsible and can be well described as a two-state transition in which the fol ded tetramer is converted directly to unfolded monomers. Increasing the pH decreases the stability of the tetramer significantly, the T-m changing fro m 341.3 K at pH 6.5 to 332.6 K at pH 9.5. The value of Delta C-p obtained f rom measurements of Delta H-m as a function of T-m was 10.7 +/- 0.7 kcal mo l(-1) K-1. The value of Delta C-p is among the highest measured for a multi meric protein. At 298 K, pH 7.4, the Delta G degrees(u) for the SecB tetram er is 27.9 +/- 2 kcal mol(-1). Denaturant-mediated unfolding of SecB was fo und to be irreversible. The reactivity of the four solvent-exposed free thi ols in tetrameric SecB is salt dependent. The kinetics of reactivity sugges ts that these four cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. Th e thermodynamic data suggest that SecB is a stable, well-folded, and tightl y packed tetramer and that substrate binding occurs at a surface site rathe r than at an interior cavity.