Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702601
Title: Roles of periplasmic chaperones and BamA in outer membrane protein folding
Author: Schiffrin, Robert
ISNI:       0000 0004 6058 3658
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
A defining feature of living things is that they have an inside and an outside, and in order for all living cells to survive, whether they are part of a blue whale or a unicellular microscopic organism, they must have mechanisms to mediate exchange with their environment. Food and energy enters the cell, and material must also leave, such as the waste products of metabolism, or virulence factors from pathogenic organisms. Lipid membranes define these boundaries, but it is membrane proteins that mediate the exchange. Although lipid bilayers can self-assemble in vitro, the assembly of complex biological membranes containing proteins requires energy and careful coordination. The work presented in this thesis examines the biogenesis pathway of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. OMPs are synthesised on cytosolic ribosomes, translocated across the inner membrane, then chaperoned across the periplasm, before folding and insertion into the OM. While OMPs can fold spontaneously into lipid membranes, this process is too slow to be biologically relevant, so a dedicated folding catalyst, the β-barrel assembly machinery (BAM) complex, is required at the OM. Recent genetic, structural and biochemical investigations have increased our understanding of OMP assembly, but key questions remain, including: How do periplasmic chaperones bind and release OMP substrates? What are the roles and interactions of BAM subunits? What is the molecular mechanism by which BAM folds and inserts OMPs? Here, an assay was developed to monitor OMP folding kinetics in vitro using intrinsic fluorescence in low concentrations of urea (0.24 M). This allowed comparison of the real-time folding behaviour of different OMPs under the same conditions for the first time (Chapter 3). The assay was then successfully extended to include OMP assembly factors, including the periplasmic chaperones Skp and SurA, and BamA, the principal component of the BAM complex, to obtain the following key results: Investigations into the interactions between Skp and OMPs of varying size (tOmpA, PagP, OmpT, OmpF and tBamA) revealed that greater Skp:OMP ratios are required to prevent the folding of 16-stranded OMPs compared with smaller 8-stranded OMPs. Supported by ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modelling and molecular dynamics simulations, the results imply a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp to protect a single substrate from aggregation (Chapter 4). Addition of further folding factors to the assay demonstrated that the model OMP tOmpA can be released and folded from its complex with Skp by BamA, possibly recapitulating an in vivo assembly pathway. BamA consists of a β-barrel membrane-embedded domain and soluble periplasmic domains, and while the release activity was shown to located in the membrane domain, the activity was greatest when full-length BamA was present. By contrast, SurA was not able to release tOmpA from Skp under the conditions employed, arguing against a sequential chaperone model (Chapter 5). Next, kinetic studies were used to investigate the mechanism of OMP folding catalysis by the BAM complex. The effect of hydrophobic mismatch between the BamA β-barrel and the membrane was examined by monitoring the folding of tOmpA into liposomes containing lipids of different chain lengths in the presence or absence of BamA. The results showed that BamA has a greater catalytic effect in lipids with longer chain lengths, with the largest rate enhancement achieved in bilayers with a hydrophobic thickness close to that of the OM. The results establish the importance of hydrophobic mismatch in the mechanism by which OMPs are folded in vivo, which may be influenced by local thinning of the membrane and increases in lipid disorder in the vicinity of the BAM complex (Chapter 5). Finally, based on the results obtained in this project, and consideration of the currently available literature, a new 'barrel elongation' model is proposed for the mechanism of OMP assembly by the BAM complex (Chapter 6). The OMP assembly pathway is an attractive target for novel antibacterials given that it is surface located, highly conserved, and essential in clinically important pathogens. Understanding the molecular mechanisms of OMP biogenesis factors will facilitate the development of drugs targeting this pathway. The work described in this thesis provides new insights into the mechanisms of OMP assembly, using a wide range of biochemical and biophysical techniques, thereby contributing to the development of this fast-moving and fascinating field.
Supervisor: Radford, Sheena E. ; Brockwell, David J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.702601  DOI: Not available
Share: