Unfolding Thermodynamics of the Tetrameric Chaperone, SecB

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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 Tm changing from 341.3 K at pH 6.5 to 332.6 K at pH 9.5. The value of ΔCp obtained from measurements of ΔHm as a function of Tm was 10.7 ± 0.7 kcal mol-1 K-1. The value of ΔCp 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.