Mechanistic Insights into Outer Membrane Protein Folding

Abstract

The beta-barrel outer membrane proteins (OMPs) of Gram-negative bacteria are essential for cell viability and survival, but the molecular mechanisms of their biogenesis remain poorly understood. OMPs are synthesised in the cytoplasm, translocated across the inner membrane and transported across the periplasm by the molecular chaperones Skp or SurA, before being inserted into the OM by the essential beta-barrel assembly machinery (BAM) complex (a 203 kDa heteropentameric membrane protein complex). The mechanisms by which Skp and SurA recognise OMPs and successfully deliver their cargos to BAM remain poorly understood. Questions about these mechanisms are especially interesting since the periplasm is devoid of ATP, and it is completely unknown how substrate binding and release are controlled and coordinated. Moreover, whilst several structures of the BAM complex have been solved recently, how OMPs are delivered to BAM from Skp/SurA and folded into the crowded outer membrane remain mysterious. We have been exploiting a range of biophysical methods to gain new insights into the mechanisms of OMP folding and assembly, and its assistance by chaperones and BAM. In this lecture I will describe our recent results using a variety of OMPs of different sizes combined with analysis using fluorescence kinetic assays, cryo-EM and non-covalent mass spectrometry. The results have revealed a new mechanism of Skp action and new insights into how BAM facilitates OMP folding into the bacterial outer membrane.