Tight hydrophobic contacts with the SecB chaperone prevent folding of substrate proteins

The molecular chaperone SecB binds to hydrophobic sections of unfolded secretory proteins and thereby prevents their premature folding prior to secretion by the translocase of Escherichia coli. Here, we have investigated the effect of the single-residue mutation of leucine 42 to arginine (L42R) centrally positioned in the polypeptide binding pocket of SecB on its chaperonin function. The mutant retains its tetrameric structure and SecA targeting function but is defective in its holdase activity. Isothermal titration calorimetry and single-molecule optical tweezer studies suggest that the SecB(L42R) mutant exhibits a reduced polypeptide binding affinity allowing for partial folding of the bound polypeptide chain rendering it translocation-incompetent.