Single molecule measurements provide insight into general secretory system activity

How are proteins transported across membranes? The general secretory (Sec) system of E. coli exports precursor proteins via a translocase comprising the peripheral ATPase SecA and the translocon, SecYEG. Structural changes of active translocases underlie the translocation process. Yet, the mechanistic details of this complex and dynamic system have proven challenging to study. The atomic force microscope (AFM) is well suited for imaging membrane proteins in near-native conditions and can achieve molecular-scale (~10 Å) lateral resolution coupled with ~1 Å vertical resolution (i.e., normal to the bilayer surface). We imaged individual components of the Sec system as well as active translocases at work in lipid bilayers. In this talk I will review our single molecule results in the context of conventional models in the field, which are based on bulk assays. In addition to imaging, AFM can be used in force spectroscopy mode, providing access to energy landscapes. Our group has applied precision AFM-based force spectroscopy techniques to study a peptide-lipid interaction underlying Sec system activity. Together with analytical modeling and simulations, the results represent a step towards a more detailed understanding of the protein export process in E. coli, and more generally, of the stochastic kinetic pathways driving peptide-lipid interactions.