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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.