| The bacterial Sec pathway is responsible for secretory protein translocation and integration of membrane imbedded proteins. The main components include a membrane embedded SecYEG translocon channel and a cytosolic SecA ATPase. SecA binds to a preprotein in the cytosol, bringing it to the translocon, and then is believed to push the preprotein through the translocon via repeated cycles of ATP binding and hydrolysis.;For a preprotein to be directed to the Sec pathway, a signal sequence needs to be recognized and bound by SecA. Previous experiments examining the signal sequence binding location on SecA lack resolution due to techniques which can only provide 2-dimensional binding sites. We sought to resolve this issue by mapping a 3-dimensional signal peptide (SP) binding site using Forster resonance energy transfer (FRET). Distance measurements determined for nine SecA mutants indicate that the SP-binding domain is located within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel component. Fluorescence anisotropy was utilized to determine equilibrium binding constants of 1.4 muM or 10.7muM for the alkaline phosphatase (PhoA) SP labeled at residue 22 or 2, respectively, for SecA, with a binding stoichiometry of one SP bound per SecA protomer. Furthermore, we determined that SP binds monomeric or dimeric SecA with comparable affinity.;We have also addressed the controversy over the solution state orientation of the E. coli SecA dimer using FRET. By measuring interprotomer distances between residues in the SecA dimer and comparing them to the distances predicted from four SecA dimer crystal structures, we were able to determine that the solution state SecA dimer is an anti-parallel dimer closely resembling the SecA dimer structure solved by Hunt et al. (Hunt, Weinkauf et al. 2002). Additionally, two PhoA SPs were employed to examine SP induced changes within dimeric SecA. Monomerization of SecA was not observed, however, there was conformational movement within the PPXD and HWD. The movement of the PPXD was further enhanced by the binding of an elongated SP containing residues from the early mature region of the preprotein. |
