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In vitro folding and remodelling of outer membrane proteins Bob Schiffrin, Sam Hickman, Sheena Radford and David Brockwell Introduction The outer membranes (OM) of Gram negative bacteria perform numerous essential and diverse functions, such as nutrient uptake (of vitamin B12 by BtuB, for example), secretion of virulence factors and the maintenance of cell integrity. These functions are mediated by β-barrel outer membrane proteins (OMPs) which are found exclusively in the OM of bacteria, mitochondria and chloroplasts. We are currently investigating two aspects of OMP folding and dynamics: (i) the folding and assembly of a range of OMPs and the role associated assembly factors and (ii) the mechanical gating of the plugged lumen of an OMP by force-induced remodelling. Figure 1: the OMP assembly pathway. Despite the availability of crystal structures for all proteins involved, very little is known at the molecular level about their role, function and interactions. Key questions include: What is the molecular mechanism by which the Bam complex folds and inserts OMPs? What is the role of individual Bam components? How do periplasmic chaperones deliver OMP substrates to the Bam complex? Results (i) The folding of OMPs in vivo is catalysed by the β-barrel Assembly Machinery (BAM) complex, a large hetero-oligomeric complex (Figure 1). BAM is an excellent potential antibiotic target given that it is essential, surface located and widely conserved across Gram negative species. Our work aims to gain insight into OMP assembly by recapitulating aspects of the Figure 2: spectroscopic measurement of OMPs folding into liposomes. (a) tOmpA: porin/adhesion, 19kDa. (b) PagP: enzyme, 19 kDa. (c) tBamA: insertase/secretase, 43 kDa. (d) BtuB: transporter, 66 kDa. All measurements carried out using 100 nm diC11:0PC liposomes, in 0.24M urea, 50 mM glycine-NaOH, pH 9.5 at 25 °C. Inserts: crystal structures of tOmpA (PDB: 1QJP), PagP (PDB: 36P6), tBamA (PDB: 4C4V) and BtuB (PDB: 2GUF). pathway using purified components. We have developed an in vitro assay to study the kinetics of chaperone-mediated folding of OMPs. We have shown that from an unfolded state in 8M urea, OMPs of different barrel sizes and structures can be folded into synthetic liposomes by rapid dilution, and the folding rate measured spectroscopically by monitoring changes in OMP intrinsic fluorescence (Figure 2). Work is now on going to dissect the effects of inclusion of folding factors in the assay on in vitro OMP folding. Using these kinetic assays, and a range of other biochemical and biophysical methods, we hope to provide new insights into the molecular mechanism of the BAM complex, chacterise its interactions with OMPs and periplasmic chaperones and assess how BAM modulates the OMP folding pathway. (ii) Gram-negative bacteria use a specialised transport system to scavenge scarce essential nutrients, and actively transport them across the outer-membrane. How the interaction between the inner-membrane protein TonB and the Ton box of TonB-dependent receptors facilitates transport, however, remains unclear (Figure 3a). Our research has utilised atomic force microscopy (Figure 3b) to demonstrate that TonB is able to remodel the globular plug domain inside the core of the β-barrel using mechanical force before the non-covalent trans-membrane complex dissociates (Figure 3c). The remodelling is believed create a channel through the Figure 3: (a) the TonB-dependent transport system, TonB spans the periplasmic space and forms a 1:1 complex with BtuB by binding to the Ton Box. (b) the AFM experimental set up for investigating the BtuB plug remodelling when extended by mechanical force. (c) example traces showing the unfolding of the plug followed by the unbinding of TonB. receptor that allows the passage of the bound substrate into the periplasmic space. Publications McMorran, L., Brockwell, D. & Radford, S. (2014) Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date? Arch. Biochem. Biophys. 564: 265-280. Funding This work was funded by the BBSRC and the Wellcome Trust.