This is the peer reviewed version of the article which has been published in final form at http://dx.doi.org/10.1002/anie.201509910. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.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.Integral β-barrel membrane proteins are the predominant structural class in the outer membranes of bacteria, chloroplasts and mitochondria.[1-4] The three-dimensional structure of a β-barrel membrane protein is defined by backbone hydrogen bonds between adjacent strands. [2,5,6] The biogenesis pathways of β-barrel membrane proteins are essential, but the underlying mechanism is still unclear. [7][8][9] In vitro, β-barrel membrane proteins can be functionally refolded from a chaotrope-denatured state into detergent micelles or lipid bilayers, ending up in their native structure. [10][11][12] Atomic resolution descriptions of in vitro folding thus provide important benchmark data for the folding biophysics and yield insight into structural determinants. In vitro folding of β-barrel membrane proteins has been characterized using biophysical and biochemical methods, including fluorescence spectroscopy, [13,14] circular dichroism (CD) spectroscopy, [15][16][17] SDS-PAGE migration shifts, [18,19] and singlemolecule force spectroscopy. [20] However, despite the fundamental role for the protein structure, the formation of the hydrogen bond network during β-barrel membrane protein folding has so far not been observed at the atomic level.Here, we monitor the formation of interstrand hydrogen bonds during the folding process of a β-barrel membrane protein in detergent micelles by hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry. [21,22] We use the 8-stranded OmpX from E. coli as a model system. [23,24] At the onset of the experiment, OmpX (148 aa) is solubilized in chaotropic denaturant solution, where the polypeptide is fully unfolded and adopts a random coil conformation without residual structure.[25-27] Using a manually operated, custom-buil...
