Thermodynamics of substrate binding to the chaperone SecB

The thermodynamics of binding of unfolded polypeptides to the chaperone Sec B was investigated in vitro by isothermal titration calorimetry and fluores cence spectroscopy. The substrates were reduced and carboxamidomethylated f orms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolde d RNase A and BPTI as well as compact, partially folded disulfide intermedi ates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heal capacity changes observed on binding the reduced and carboxamidom ethylated forms of cr-lactalbumin, BPTI, and RNase A were found to be -0.10 , -0.29, and -0.41 kcal mol(-1) K-1, respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer o f SecB. There is no evidence for two separate types of binding sites for po sitively charged and hydrophobic ligands. Spectroscopic and proteolysis pro tection studies of the binding of SecB to poly-L-Lys show that binding of h ighly positively charged peptide ligands to negatively charged SecB leads t o charge neutralization and subsequent aggregation of SecB,The data are con sistent with a model where SecB binds substrate molecules at an exposed hyd rophobic cleft. SecB aggregation in the absence of substrate is prevented b y electrostatic repulsion between negatively charged SecB tetramers.