title: Monitoring Backbone Hydrogen-Bond Formation in β-Barrel Membrane Protein Folding.
abstract: β-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire β-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale.
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 70, 4056, Basel, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Raschle
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 70, 4056, Basel, Switzerland.|Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, Mattenstrasse 26, 4058, Basel, Switzerland.
authors.forename: Perla
authors.initials: P
authors.lastname: Rios Flores
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 70, 4056, Basel, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Opitz
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, Mattenstrasse 26, 4058, Basel, Switzerland.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Müller
authors.affiliation: Biozentrum, University of Basel, Klingelbergstrasse 70, 4056, Basel, Switzerland. sebastian.hiller@unibas.ch.
authors.forename: Sebastian
authors.identifier: 0000-0002-6709-4684
authors.initials: S
authors.lastname: Hiller
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D012997:Solvents; C482905:OmpX protein, E coli; D006867:Hydrolases
keywords: NMR spectroscopy; fluorescence spectroscopy; mass spectrometry; membrane proteins; protein folding