SecA folds via a dimeric intermediate

Though many proteins in the cell are large and multimeric, their folding ha s not been extensively studied. We have chosen SecA as a folding model beca use it is a large, homodimeric protein (monomer molecular mass of 102 kDa) with multiple folding domains. SecA is the ATPase for the Sec-dependent pre protein translocase of many bacteria. SecA is a soluble protein that can pe netrate into the membrane during preprotein translocation. Because SecA may partially unfold prior to its insertion into the membrane, studies of its stability and folding pathway are important for understanding how it functi ons in vivo. Kinetic folding transitions in the presence of urea were monit ored using circular dichroism and tryptophan fluorescence, while equilibriu m folding transitions were monitored using the same techniques as well as a fluorescent ATP analogue. The reversible equilibrium folding transition ex hibited a plateau, indicating the presence of an intermediate. Based on the data presented here, we propose a three-state model, N2 double left right arrow I-2 double left right arrow 2U, where the native protein unfolds to a dimeric intermediate which then dissociates into two unfolded monomers. Th e SecA dimer was determined to have an overall stability (Delta G) of -22.5 kcal/ mel. We also investigated the stability of SecA using analytical ult racentrifugation equilibrium and velocity sedimentation, which again indica ted that native or refolded SecA was a stable dimer. The rate-limiting step in the folding pathway was conversion of the dimeric intermediate to the n ative dimer. Unfolding of native, dimeric SecA was slow with a relaxation t ime in H2O of 3.3 x 10(4) s. Since SecA is a stable dimer, dissociation to monomeric subunits during translocation is unlikely.