Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB

SecB is a homotetrameric, cytosolic chaperone that forms part of the protei n translocation machinery in Escherichia coli. We have investigated the bou nd-state conformation of a model protein substrate of SecB, bovine pancreat ic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by u sing proximity relationships based on site-directed spin-labeling and pyren e fluorescence methods. BPTI is a 58-residue protein and contains three dis ulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. M utants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyre ne maleimide. The relative proximity of the labeled residues was studied us ing either electron spin resonance spectroscopy or fluorescence spectroscop y. The data suggest that SecB binds a collapsed coil of reduced unfolded BP TI, which then undergoes a structural rearrangement to a more extended stat e upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substra te residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The elect ron spin resonance data suggest that these cysteine residues are in close p roximity (15 Angstrom) when no substrate protein is bound but move away to a distance of greater than 20 Angstrom when SecB binds substrate. This is t he first direct evidence of a conformational change in SecB upon binding of a substrate protein.