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Surface-tethered planar membranes containing the ß-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding.
Hall, Stephen C L; Clifton, Luke A; Sridhar, Pooja; Hardy, David J; Wotherspoon, Peter; Wright, Jack; Whitehouse, James; Gamage, Nadisha; Laxton, Claire S; Hatton, Caitlin; Hughes, Gareth W; Jeeves, Mark; Knowles, Timothy J.
Affiliation
  • Hall SCL; ISIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxfordshire, United Kingdom.
  • Clifton LA; ISIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxfordshire, United Kingdom.
  • Sridhar P; School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
  • Hardy DJ; School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
  • Wotherspoon P; School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
  • Wright J; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
  • Whitehouse J; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
  • Gamage N; Membrane Protein Laboratory, Diamond Light Source, Harwell Science & Innovation Campus, Oxfordshire, United Kingdom.
  • Laxton CS; School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
  • Hatton C; School of Life Sciences, University of Warwick, Coventry, United Kingdom.
  • Hughes GW; School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
  • Jeeves M; Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom.
  • Knowles TJ; School of Biosciences, University of Birmingham, Birmingham, United Kingdom. Electronic address: t.j.knowles@bham.ac.uk.
Biophys J ; 120(23): 5295-5308, 2021 12 07.
Article in En | MEDLINE | ID: mdl-34757080
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 ß-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 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, 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.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Escherichia coli Proteins Language: En Journal: Biophys J Year: 2021 Document type: Article Affiliation country: Country of publication:

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Escherichia coli Proteins Language: En Journal: Biophys J Year: 2021 Document type: Article Affiliation country: Country of publication: