Research Data Leeds Repository

Supporting data for “The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding”

White, Paul and Haysom, Samuel and Iadanza, Matthew G. and Higgins, Anna J. and Machin, Jonathan and Whitehouse, James and Carpenter-Platt, Charlotte and Horne, Jim E. and Brockwell, David J. and Ranson, Neil A. and Radford, Sheena E. (2021) Supporting data for “The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding”. University of Leeds. [Dataset] https://doi.org/10.5518/835

Dataset description

The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the BamA β-barrel, the core component of BAM, catalyses OMP folding remains unclear, with asymmetry in the depth of BamA potentially playing a role by disrupting membrane stability. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite all being lethal in vivo, we show that all catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM revealed that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes destabilise the lipid bilayer, while BamA alone does not, highlighting the requirement of the BAM lipoproteins for this function. Together the results provide new insights into the role of BAM structure and lipid dynamics in OMP folding

Additional information:	*** This dataset and associated paper are still under review and may be changed. ***
Keywords:	Outer membrane protein, BAM, membrane, lipid, folding, structure, CryoEM
Subjects:	C000 - Biological sciences > C700 - Molecular biology, biophysics & biochemistry
Divisions:	Faculty of Biological Sciences > School of Molecular and Cellular Biology Faculty of Biological Sciences > Biomedical and Health Research Centre Faculty of Engineering and Physical Sciences > Biomedical and Health Research Centre Faculty of Engineering and Physical Sciences > Biomedical and Health Research Centre Faculty of Medicine and Health > Biomedical and Health Research Centre
License:	Creative Commons Attribution 4.0 International (CC BY 4.0)
Date deposited:	02 Jun 2021 08:33
URI:	http://archive.researchdata.leeds.ac.uk/id/eprint/841
Additional details Additional details Creators: Creators ORCID Other ID Email Primary affiliation Primary affiliation ID Primary affiliation ID type Secondary affiliation Secondary affiliation ID Secondary affiliation ID type White, Paul https://orcid.org/0000-0001-7536-4552 University of Leeds https://ror.org/024mrxd33 ROR Haysom, Samuel https://orcid.org/0000-0002-8769-090X University of Leeds https://ror.org/024mrxd33 ROR Iadanza, Matthew G. https://orcid.org/0000-0001-6152-1844 University of Leeds https://ror.org/024mrxd33 ROR Higgins, Anna J. https://orcid.org/0000-0001-8407-950X University of Leeds https://ror.org/024mrxd33 ROR Machin, Jonathan https://orcid.org/0000-0002-1920-149X University of Leeds https://ror.org/024mrxd33 ROR Whitehouse, James https://orcid.org/0000-0003-0088-4312 University of Leeds https://ror.org/024mrxd33 ROR Carpenter-Platt, Charlotte University of Leeds https://ror.org/024mrxd33 ROR Horne, Jim E. https://orcid.org/0000-0001-5260-2634 University of Leeds https://ror.org/024mrxd33 ROR Brockwell, David J. https://orcid.org/0000-0002-0802-5937 University of Leeds https://ror.org/024mrxd33 ROR Ranson, Neil A. https://orcid.org/0000-0002-3640-5275 University of Leeds https://ror.org/024mrxd33 ROR Radford, Sheena E. https://orcid.org/0000-0002-3079-8039 University of Leeds https://ror.org/024mrxd33 ROR Type of data: Dataset Research funders: Medical Research Council UK, BBSRC, Wellcome Trust, Wellcome Trust, FWO and FRS-FNRS, Royal Society Grant numbers: MR/P018491/1, BB/M011151/1, 105220/Z/14/Z, 222373/Z/21/Z, G0G0818N, RSRP/R1/211057 Publication date: 2021 Resource language: English Metadata language: English Publisher: University of Leeds Contact Email Address: bssfh@leeds.ac.uk Last Modified: 23 Jan 2023 16:34

Files

Documentation

Readme file [9kB] Readme file [9kB] Readme file [9kB] Visible to: Anyone File name: README.txt Content type: Documentation Description: Description of dataset and instructions on how to navigate the data Mime-Type: text/plain License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 9kB

List of files [23kB] List of files [23kB] List of files [23kB] Visible to: Anyone File name: file_list.txt Content type: Documentation Mime-Type: text/plain License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 23kB

Data

DLS Data [567B] DLS Data [567B] DLS Data [567B] Visible to: Anyone File name: DLS.zip Content type: Data Description: Dynamic light scattering data Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 567B

Kinetics data [437kB] Kinetics data [437kB] Kinetics data [437kB] Visible to: Anyone File name: Kinetics.zip Content type: Data Description: Quantification of kinetics assays monitoring folding of small 8-stranded OMPs by band shift on SDS-PAGE gels Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 437kB

Laurdan data [52kB] Laurdan data [52kB] Laurdan data [52kB] Visible to: Anyone File name: Laurdan.zip Content type: Data Description: Raw data obtained for laurdan membrane destabilisation assay Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 52kB

OMP folding gels [308MB] OMP folding gels [308MB] OMP folding gels [308MB] Visible to: Anyone File name: OMP_Folding_Gels.zip Content type: Data Description: Raw gels underlying kinetics data Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 308MB

Proteoliposome Quality control gels [25MB] Proteoliposome Quality control gels [25MB] Proteoliposome Quality control gels [25MB] Visible to: Anyone File name: Proteoliposome_QC_gels.zip Content type: Data Description: Raw gels from the preparation process for the BAM containing proteoliposomes used in folding assays Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 25MB

Proteoliposome trypsin digest gels [700kB] Proteoliposome trypsin digest gels [700kB] Proteoliposome trypsin digest gels [700kB] Visible to: Anyone File name: Proteoliposome_trypsin_digest.zip Content type: Data Description: Raw gels from the trypsin digestion of BAM containing proteoliposomes carried out to test the orientation of BAM Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 700kB

Thiol labelling fluorescence gel [2MB] Thiol labelling fluorescence gel [2MB] Thiol labelling fluorescence gel [2MB] Visible to: Anyone File name: thiol_labelling_fluorescence_gel.zip Content type: Data Description: Raw gels for the thiol labelling gel of the disulphide locked BAM variants to assess degree of disulphide formation Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 2MB

Mass spectra for labelling experiments of BAM-LL [113kB] Mass spectra for labelling experiments of BAM-LL [113kB] Mass spectra for labelling experiments of BAM-LL [113kB] Visible to: Anyone File name: thiol_labelling_MS.zip Content type: Data Mime-Type: application/zip License: Creative Commons Attribution 4.0 International (CC BY 4.0) File size: 113kB