Author's personal copy pH-Induced Conformational Change of the β-Barrel-Forming Protein OmpG Reconstituted into Native E. coli Lipids Stefania A. Mari 1,2 , Stefan Köster 3 , Christian A. Bippes 1,2 , Özkan Yildiz 3 , Werner Kühlbrandt 3 and Daniel J. Muller 1,2 ⁎ 1 Biotechnology Center, University of Technology, Tatzberg 47, 01307 Dresden, Germany 2 Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland 3 Max-Planck-Institute of Biophysics, Max-von-Laue- Straße 3, 60438 Frankfurt am Main, Germany Received 16 October 2009; received in revised form 11 December 2009; accepted 17 December 2009 Available online 28 December 2009 A gating mechanism of the β-barrel-forming outer membrane protein G (OmpG) from Escherichia coli was recently presented. The mechanism was based on X-ray structures revealed from crystals grown from solubilized OmpG at both neutral pH and acidic pH. To investigate whether these conformations represent the naturally occurring gating mechanism, we reconstituted OmpG in native E. coli lipids and applied high-resolution atomic force microscopy. The reconstituted OmpG molecules assembled into both monomers and dimers. Single monomeric and dimeric OmpG molecules showed open channel entrances at pH 7.5 and at room temperature. The extracellular loops connecting the β-strands that form the transmembrane β-barrel pore exhibited elevated structural flexibility. Upon lowering the pH to 5.0, the conformation of OmpG molecules changed to close the extracellular entrance of their channel. It appears that one or more of the extracellular loops collapsed onto the channel entrance. This conformational change was fully reversible. Our data confirm that the previously reported gating mechanism of OmpG occurs at physiological conditions in E. coli lipid membranes. © 2009 Elsevier Ltd. All rights reserved. Edited by W. Baumeister Keywords: atomic force microscopy; β-barrel membrane protein; Escherichia coli; interactions; dimers Introduction The gating mechanism of outer membrane proteins (Omps) attracts continuous interest and remains to be explained in detail. 1 Some of the transmembrane pores formed by Omps of Escherichia coli are pH gated. Low pH induces the closing of the channels of, for example, OmpC, OmpF, OmpG, LamB and PhoE. 2–6 What conformational change causes channel closure has long been debated. 1,7 In 1999, atomic force microscopy (AFM) for the first time showed that the large extracellular loops of OmpF collapsed onto the channel entrance at low pH or upon reaching a critical voltage. 8 This supported the theory that conforma- tional changes of the flexible extracellular loops could gate Omp channels. Experiments on maltoporin (LamB), an E. coli β-barrel-forming porin that is specific for malto-oligosaccharides, corroborated the gating model. LamB lacking the major extracellular loops L4 and L6 failed to close at lower pH. 9 However, due to the lack of high-resolution struc- tures, as revealed by X-ray crystallography, these insights could not be further substantiated. Further- more, it remains to be shown whether conformational changes of flexible extramembranous polypeptide loops are a common gating mechanism of β-barrel- forming Omps. Recently, the structure of OmpG from E. coli was solved by X-ray crystallography 10,11 and NMR. 12 OmpG comprises 14 β-strands that form a transmem- brane β-barrel. Six short polypeptide turns (T1–T6) connect β-strands on the periplasmic side. Seven longer loops (L1–L7) that exhibit enhanced intrinsic flexibility 11,12 connect β-strands on the extracellular side. Being able to withstand rather harsh environ- mental conditions, OmpG has potential to be *Corresponding author. Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland. E-mail address: daniel.mueller@bsse.ethz.ch. Abbreviations used: Omp, outer membrane protein; AFM, atomic force microscopy. doi:10.1016/j.jmb.2009.12.034 J. Mol. Biol. (2010) 396, 610–616 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.