Digestion of vesicle-bound SecA by trypsin entrapped within the vesicles showed that refolding precursor ribose-binding protein (pRBP) of Escherichia coli retards the lipid bilayer penetration by SecA while the signal peptide enhances it. This discrepancy was found to be due to reduced SecA binding to the vesicles in the presence of the pRBP while the signal peptide induced a tight binding. Studies on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to SecA indicated that SecA assumes more closed conformation upon interaction with pRBP and signal peptide induces more open structure of SecA. Kinetic studies of ANS binding to SecA upon dilution of unfolded pRBP with SecA solution showed an initial fast ANS binding, which was followed by a slow release of ANS. This suggests that first the signal peptide portion of the pRBP binds with the SecA making its structure more open and then the subsequent binding of the mature domain makes the SecA structure more compact. The pRBP enhanced the digestion of SecA added to the E. coli inverted vesicles, suggesting an inhibition of SecA penetration while the signal peptide had an opposite effect, agreeing with the results from the model systems above. When the pRBP and ATP were present together, however, the penetration of SecA increased dramatically underlining the importance of the SecY/E complex for the membrane insertion of SecA.

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