Surface-tethered planar membranes containing the β-barrel assembly machinery: A platform for investigating bacterial outer membrane protein folding

DOI: 10.1016/j.bpj.2021.10.033

Authors: Stephen C. I. Hall (ISIS Pulsed Neutron and Muon Source) , Luke Clifton (ISIS Pulsed Neutron and Muon Source) , Pooja Sridhar (University of Birmingham) , David J. Hardy (University of Birmingham) , Peter Wotherspoon (University of Birmingham) , Jack Wright (University of Leeds) , James Whitehouse (University of Leeds) , Nadisha Gamage (Diamond Light Source) , Claire S. Laxton (University of Nottingham) , Caitlin Hatton (University of Warwick) , Gareth W. Hughes (University of Birmingham) , Mark Jeeves (University of Birmingham) , Timothy J. Knowles (University of Birmingham)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biophysical Journal

State: Published (Approved)
Published: October 2021

Abstract: The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry (PNR) revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials

Facility: ISIS

Added On: 02/11/2021 10:37

Discipline Tags:

Biophysics Life Sciences & Biotech

Technical Tags: