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The Ins and Outs of Membrane Proteins

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The Ins and Outs of Membrane Proteins The Ins and Outs of Membrane Proteins Topology Studies of Bacterial Membrane Proteins Mikaela Rapp Department of Biochemistry and Biophysics Stockholm University, Sweden 2006 © Mikaela Rapp, Stockholm 2006 ISBN 91-7155-311-8, pages 1-58 All previously published articles are reprinted with permission from the publishers Typesetting: Intellecta Docusys Printed in Sweden by Intellecta Docusys, Stockholm 2006 Distributor: Stockholm University Library Till min familj Abstract α-helical membrane proteins comprise about a quarter of all proteins in a cell and carry out a wide variety of essential cellular functions. This thesis is focused on topology analyses of bacterial membrane proteins. The topology describes the two-dimensional structural arrangement of a protein relative to the membrane. By combining large-scale experimental and bioinformatics techniques we have produced experimentally constrained topology models for the major part of the Escherichia coli membrane proteome. This represents a substantial increase in available topology information for bacterial membrane proteins. Many membrane protein structures show signs of internal duplication and approximate two-fold in-plane symmetry. We propose a step-wise pathway to explain how proteins with such internal inverted repeats have evolved. The pathway is based on the ‘positive-inside’ rule and starts with a protein that can adopt two topologies in the membrane, i.e. a “dual” topology protein. The gene encoding the dual topology protein is duplicated and eventually, through re-distribution of positively charge residues, the two resulting homologous proteins become fixed in opposite orientations in the membrane. Finally, the two proteins may fuse into one single polypeptide with an internal inverted repeat structure. Finally, we re-create the proposed step-wise evolutionary pathway in the laboratory by showing that only a small number of mutations are required in order to transform the homo-dimeric, dual topology protein EmrE into a hetero-dimeric complex composed of two oppositely oriented proteins. Contents List of Publications.............................................................................................. 1 Abbreviations ...................................................................................................... 2 Introduction ......................................................................................................... 3 The biological membrane ............................................................................................. 4 Biogenesis of MPs........................................................................................................ 5 An overview of the “birth and life” of MPs ..................................................................... 6 The two-stage model .................................................................................................... 7 Membrane Protein Topology ............................................................................. 9 Hydrophobicity.............................................................................................................. 9 An introduction to the thermodynamic view of insertion and α -helix formation......... 9 The α-helix formation-site...................................................................................... 10 Length and hydrophobicity of TMHs ...................................................................... 10 Cracking the insertion code................................................................................... 10 The membrane-water interface region........................................................................ 12 Loops and tails ...................................................................................................... 12 The positive-inside rule ......................................................................................... 13 The ‘snorkel’ effect ................................................................................................ 16 The aromatic belt .................................................................................................. 16 Topogenic determinants work in concert .................................................................... 18 What contributes to the orientation of the signal sequence?.................................. 18 Helical hairpins...................................................................................................... 18 A joint translocon/lipid system deciphers the unique topology of a MP.................. 19 The evolution of membrane proteins .......................................................................... 20 Internal repeats ..................................................................................................... 20 Internal inverted repeats........................................................................................ 20 Membrane Protein Structure............................................................................ 21 Intrahelical interactions ............................................................................................... 21 α-Helical distortions............................................................................................... 21 Interhelical interactions ............................................................................................... 22 Attractive forces that may influence MP structure formation.................................. 22 Topological constraints............................................................................................... 24 The role of interhelical loops in the folding and stability of MPs............................. 24 The role of the helical backbone dipolar moment in packing of MPs ..................... 24 Re-entrant loops.................................................................................................... 25 Helix packing models.................................................................................................. 26 Knobs-into-holes (left handed crossing angles)..................................................... 26 The GxxxG motif (right handed crossing angles)................................................... 26 Protein-Lipid interactions ............................................................................................ 27 Curvature stress.................................................................................................... 27 Hydrophobic mismatch.......................................................................................... 27 Specific protein-lipid interactions ........................................................................... 28 Lipid-dependent topogenesis of multi-spanning MPs ............................................ 28 Escherichia coli as a Model Organism............................................................ 29 Cell architecture.......................................................................................................... 29 The K-12 genome....................................................................................................... 29 Experimental Approaches to Reveal MP Structure........................................ 31 3D structural models................................................................................................... 31 Topology mapping ...................................................................................................... 31 The C-tail and the internal loops: main sites for experimental topology mapping .. 32 Reporter fusions.................................................................................................... 32 Bioinformatics - a Theoretical Approach to Study MPs ................................. 33 Sequence comparisons of proteins............................................................................. 33 BLAST................................................................................................................... 33 Pfam...................................................................................................................... 34 Prediction methods..................................................................................................... 34 Topology prediction methods ................................................................................ 34 Summary of Papers I and II ............................................................................. 39 Large-scale topology analysis .................................................................................... 39 Experimentally based topology models for E. coli inner MPs (Paper I).................. 39 Global topology analyses of the E. coli membrane proteome (Paper II) ................ 40 Summary of Papers II, III and IV ..................................................................... 41 How did proteins with internal inverted repeats evolve? ............................................. 41 A step-wise evolutionary path for internal inverted repeats......................................... 41 Identification and evolution of dual-topology proteins (Paper III) ........................... 42 Emulating membrane protein evolution by rational design (Paper IV) ................... 43 Discussion .................................................................................................................. 44 Acknowledgements .......................................................................................... 45 References.......................................................................................................
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