CPLC Seminar: Gavin King "Single molecule measurements of topography and force reveal mechanisms underlying general secretory system activity"

10/19/2018 4:46:03 PM CPLC Staff

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. Precursor- and nucleotide-mediated structural changes of active translocases underlie the translocation process. Yet, determining mechanistic details of this complex system has proven to be challenging. 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 translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation. Topography results showed that starting from nearly identical initial states, active translocases evolve to exhibit precursor-dependent conformations at the plateau stage of activity. These data, acquired in near-native bilayers, suggest that the translocation mechanism varies with precursor species, a result that is not accounted for in conventional models of translocation. In addition to imaging, AFM can be used in force spectroscopy mode, providing access to energy landscapes. Our laboratory has developed precision AFM-based force spectroscopy techniques and applied them to study a peptide-lipid interaction that is essential to Sec system activity. Together with analytical modeling and simulations, the results represent a step towards a more detailed understanding of the export process in E coli., and more generally, of the stochastic kinetic pathways driving peptide-lipid interactions.