A thermodynamic coupling mechanism for the disaggregation of a model peptide substrate by chaperone SecB

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated s tate(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precur sor protein translocation machinery in Escherichia coil, We have investigat ed the interaction of the B-chain of insulin with chaperone SecB by light s cattering, pyrene excimer fluorescence, and electron spin resonance spectro scopy. We show that SecB prevents aggregation of the B-chain of insulin. We show that SecB is capable of dissociating soluble B-chain aggregates as mo nitored by pyrene fluorescence spectroscopy. The kinetics of dissociation o f the B-chain aggregate by SecB has been investigated to understand the mec hanism of dissociation. The data suggests that SecB does not act as a catal yst in dissociation of the aggregate to individual B-chains, rather it bind s the small population of free B-chains with high affinity, thereby shiftin g the equilibrium from the ensemble of the aggregate toward the individual B-chains, Thus SecB can rescue aggregated, partially folded/misfolded state s of target proteins by a thermodynamic coupling mechanism when the free en ergy of binding to SecB is greater than the stability of the aggregate, Pyr ene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B chain to chaperone SecB, The data su ggests that the B chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximat ely 10 residues of substrate.