Escherichia coliexports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.
AFM nanoindentation reveals decrease of elastic modulus of lipid bilayers near freezing point of water
