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Dual-Topology Membrane Proteins in Escherichia Coli Dual-topology membrane proteins in Escherichia coli Susanna Seppälä ©Susanna Seppälä, Stockholm 2011 ISBN 978-91-7447-351-3, pp. 1-66 Printed in Sweden by US-AB, Stockholm 2011 Distributor: Department of Biochemistry and Biophysics, Stockholm University ii Vanhemmilleni iii List of publications Primary publications I Rapp M*, Granseth E*, Seppälä S, von Heijne G (2006): Identification and evolution of dual-topology membrane proteins. Nature Structural and Molecular Biology 13, 112-116 II Rapp M*, Seppälä S*, Granseth E, von Heijne G (2007): Emulating membrane evolution by rational design. Science 315, 1282-1284 III Seppälä S, Slusky JS, Lloris-Garcerá P, Rapp M, von Heijne G (2010): Control of membrane topology by a single C-terminal residue. Science 328, 1698-1700 IV Lloris-Garcerá P, Bianchi F, Slusky JSG, Seppälä S, Daley DO, von Heijne G (201x): Antiparallel dimers of the small multidrug-resistance protein EmrE are more stable than parallel dimers. Manuscript in preparation (* these authors contributed equally) Additional publications Granseth E, Seppälä S, Rapp M, Daley DO, von Heijne G (2007): Membrane protein structural biology – how far can the bugs take us? (Review) Molecular Membrane Biology 24, 329-332 Xie K, Hessa T, Seppälä S, Rapp M, von Heijne G, Dalbey R (2007): Features of transmembrane segments that promote the lateral release from the translocase into the lipid phase. Biochemistry 46, 15153-15161 Cassel M, Seppälä S, von Heijne G (2008): Confronting fusion-protein based membrane protein topology mapping with reality: the Escherichia coli ClcA H+/Cl- exchange transporter. Journal of Molecular Biology 381, 860- 866 iv Abstract Cellular life, as we know it, is absolutely dependent on biological membranes; remarkable superstructures made of lipids and proteins. For example, all living cells are surrounded by at least one membrane that protects the cell and holds it together. The proteins that are embedded in the membranes carry out a wide variety of key functions, from nutrient uptake and waste disposal to cellular respiration and communication. In order to function accurately, any integral membrane protein needs to be inserted into the cellular membrane where it belongs, and in that particular membrane it has to attain its proper structure and find partners that might be required for proper function. All membrane proteins have evolved to be inserted in a specific overall orientation, so that e.g. substrate-binding parts are exhibited on the ‘right side’ of the membrane. So, what determines in which way a membrane protein is inserted? Are all membrane proteins inserted just so? The focus of this thesis is on these fundamental questions: how, and when, is the overall orientation of a membrane protein established? A closer look at the inner membrane proteome of the familiar gram-negative bacterium Escherichia coli revealed a small group of proteins that, oddly enough, seemed to be able to insert into the membrane in two opposite orientations. We could show that these dual-topology membrane proteins are delicately balanced, and that even the slightest manipulations make them adopt a fixed orientation in the membrane. Further, we show that these proteins are topologically malleable until the very last residue has been synthesized, implying interesting questions about the topogenesis of membrane proteins in general. In addition, by looking at the distribution of homologous proteins in other organisms, we got some ideas about how membrane proteins might evolve in size and complexity. Structural data has revealed that many membrane bound transporters have internal, inverted symmetries, and we propose that perhaps some of these proteins derive from dual-topology ancestors. v Table of Contents List of publications ...................................................................................... iv Abstract ......................................................................................................... v Abbreviations .............................................................................................viii Introduction................................................................................................... 9 The model organism........................................................................... 10 Biological membranes ................................................................................ 12 Membrane lipids................................................................................. 13 The lipid bilayers of E. coli ................................................................ 14 The outer lipid bilayer........................................................... 14 The inner lipid bilayer........................................................... 15 Membrane proteins............................................................................. 15 Membrane protein topology.................................................. 16 β-barrel membrane proteins ................................................. 17 α-helical membrane proteins................................................ 18 E. coli membrane proteins.................................................................. 18 Outer membrane proteins ..................................................... 18 Inner membrane proteins...................................................... 19 α-helical membrane proteins: topology, structure and evolution.......... 20 Topology and structure of α-helical bundles ..................................... 20 Structural repeats and evolution........................................... 21 Unusual topologies ............................................................................. 23 Bona fide dual-topology membrane proteins........................ 23 Other modes of dual topology............................................... 27 Biogenesis of α-helical membrane proteins.............................................. 28 Targeting and integration of membrane proteins in E. coli................ 29 vi Targeting of exported proteins.............................................. 29 Targeting of cytoplasmic membrane proteins....................... 29 The Sec translocon................................................................ 30 YidC....................................................................................... 31 Topogenesis........................................................................................ 32 The nature of the polypeptide chain ................................................... 32 Hydrophobicity and aromatic amino acid residues.............. 32 Charged amino acid residues ............................................... 33 The importance of context: neighbouring helices................. 35 Role of the translocon/insertase.......................................................... 36 The size of the protein-conducting channel .......................... 36 The surrounding membrane................................................................ 37 The effect of lipids................................................................. 37 Protein content of the membrane.......................................... 37 Methods and publications .......................................................................... 39 The model protein............................................................................... 39 Major experimental methods.............................................................. 40 Topology mapping using reporter proteins .......................... 40 Protein expression and selective radiolabelling................... 41 In vivo ethidium toxicity assays............................................ 42 Blue-Native PAGE ................................................................ 43 Cysteine labelling and crosslinking ...................................... 43 Summary of papers............................................................................. 44 Paper I................................................................................... 44 Paper II ................................................................................. 44 Paper III................................................................................ 45 Paper IV ................................................................................ 45 Conclusions and perspectives .................................................................... 47 Populärvetenskaplig sammanfattning på svenska................................... 49 Acknowledgements ..................................................................................... 50 References.................................................................................................... 52 vii Abbreviations CL cardiolipin cryo-EM cryo-electron microscopy C-terminus carboxy-terminus GFP green fluorescent protein IPTG isopropyl β-D-thiogalactopyranoside N-terminus amino-terminus SMR small multidrug resistance PCC protein-conducting channel PE phosphatidylethanolamine PG phosphatidylglycerol PhoA alkaline phosphatase RNC ribosome:nascent chain complex SRP signal recognition particle Amino acids A Ala Alanine C Cys Cysteine D Asp Aspartate E Glu Glutamate F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine viii Introduction Cellular identity relies on the existence of the cellular membrane, a semipermeable barrier that encloses
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