title: FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins.
abstract: Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts. Graphical Abstract ᅟ.
authors.affiliation: Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Watkinson
authors.affiliation: Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Antonio N
authors.initials: AN
authors.lastname: Calabrese
authors.affiliation: Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: James R
authors.initials: JR
authors.lastname: Ault
authors.affiliation: Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK. s.e.radford@leeds.ac.uk.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.affiliation: Astbury Center for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK. a.e.ashcroft@leeds.ac.uk.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005960:Glucosides / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000737:chemistry*; D055668:Photochemical Processes; D010782:Photolysis; D011108:Polymers / Q000737:chemistry*; D011437:Propylamines / Q000737:chemistry*; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D008823:Micelles; D011108:Polymers; D011437:Propylamines; C499017:amphipol A8-35; C499588:ompT protein, E coli; D017665:Hydroxyl Radical; C040358:dodecyl maltoside; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases
keywords: Amphipols; Detergents; Fast photochemical oxidation; Membrane proteins; Structural proteomics