Single Molecule Studies of the General Secretory System

Abstract

In bacteria and archaea many proteins use the protein conducting channel SecYEG either to transport across the membrane bilayer or to integrate into the bilayer. Further, it is known that the ATPase SecA binds SecYEG to perform translocation. Crystal structures of detergent-solubilized SecYEG and SecA bound to SecYEG have been reported [Nature 427, 36 (2004); Nature 455, 936 (2008)]. In recent years atomic force microscopy (AFM) has emerged as an important complementary tool to study membrane proteins at the single molecule level in near native conditions. In this work we study two central components of the bacterial secretory system (SecYEG and SecA) in membrane via AFM. We have obtained images of proteoliposomes containing just SecYEG, and SecYEG proteoliposomes assembled in the presence of SecA (SecY·A). All samples were adsorbed on mica surfaces and imaged in aqueous buffer solution. We collected several hundred images of each sample to provide statistics. Heights of SecYEG and SecY·A protruding above the lipid bilayer are in close agreement with crystal structure data and the topological asymmetry of SecYEG allows orientation determination. From volume calculations we are able to differentiate SecYEG monomers from dimers and higher order oligomeric states. Images of SecA bound to lipid (i.e, in the absence of SecYEG) were also obtained. In this case, the heights of SecA bound to the lipid are significantly different than the heights of SecY·A suggesting distinct binding modes of SecA to lipid compared to SecA to SecYEG. Further experiments and analysis will be required to conclusively determine the oligomeric state of active SecYEG during translocation.