! "#$ #"%"" &' ( ) * ( +# % , % %& - . % ( /%0 ( - - ( 1 2 3 3 (1 2 1 2 . 4# % . - - % "#$ 566 %% 6 7 8 55 5 5 #9 : ; 0<;$:;#$$;$"" 0<;$:;#$$;$""== (#" ;# INSERTION STUDIES OF MODEL TRANSMEMBRANE SEGMENTS INTO BACTERIAL AND EUKARYOTIC MEMBRANES Nina Schiller Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes Nina Schiller ©Nina Schiller, Stockholm University 2017 ISBN print 978-91-7797-002-6 ISBN PDF 978-91-7797-003-3 Printed in Sweden by Universitetsservice US-AB, Stockholm 2017 Distributor: Department of Biochemistry and Biophysics, Stockholm University To my family Contents List of publications ........................................................................................... 1 Additional publications .................................................................................... 2 Abstract ........................................................................................................... 3 Sammanfattning på svenska ........................................................................... 4 Introduction ..................................................................................................... 5 1 The prokaryotic cell ................................................................................ 7 2 The eukaryotic cell ................................................................................. 9 3 Biological membranes ......................................................................... 11 3.1 General features of biological membranes ....................................................... 11 3.2 Different types of biological membranes ........................................................... 12 3.3 The bacterial membrane ................................................................................... 14 3.4 The endoplasmic reticulum ............................................................................... 15 4 Proteins inside or at the surface of the membrane .............................. 17 4.1 ȕ-barrel membrane proteins ............................................................................. 19 4.2 Į-helical membrane proteins ............................................................................ 19 4.3 Protein-lipid interactions ................................................................................... 20 5 Protein biogenesis ............................................................................... 22 5.1 Protein synthesis within the ribosome............................................................... 22 6 Protein targeting and transport ............................................................ 24 6.1 Signal sequence ............................................................................................... 24 6.2 Protein targeting to the bacterial inner membrane and the ER ......................... 25 6.2.1 The co-translational protein targeting pathway ...................................... 26 6.2.2 Post-translational protein targeting pathway .......................................... 27 7 Protein translocation machineries ........................................................ 29 7.1 The Sec translocon .......................................................................................... 29 7.2 Accessory components .................................................................................... 31 7.2.1 Bacterial SecDFYajC and YidC.............................................................. 31 7.2.2 Eukaryotic TRAM, Calnexin, Calreticulin, OST, PDI .............................. 31 7.3 Signal peptidase ............................................................................................... 33 8 ,QWHJUDWLRQRIĮ-helical membrane proteins into the membrane .......... 34 9 3URSHUWLHVRIĮ-helical transmembrane segments ............................... 35 9.1 Polar amino acids in transmembrane segments ............................................... 35 9.2 Proline and glycine in transmembrane segments ............................................. 36 9.3 The “aromatic belt” ........................................................................................... 36 9.4 “Snorkeling” ...................................................................................................... 36 10 Membrane protein topology ................................................................. 38 10.1 The positive inside rule ............................................................................... 39 10.2 Lipids as topology determinants .................................................................. 40 10.3 Other topology determinants ....................................................................... 40 10.4 Hydrophobic mismatch ................................................................................ 41 10.5 Re-entrant regions ...................................................................................... 41 10.6 Topology mapping....................................................................................... 41 10.6.1 PhoA ...................................................................................................... 41 10.6.2 GFP ....................................................................................................... 42 10.6.3 Glycosylation mapping ........................................................................... 42 10.6.4 Protease-protection assay ..................................................................... 42 10.6.5 Epitope tags ........................................................................................... 42 10.7 Topology predictions ................................................................................... 43 10.7.1 Hydrophobicity scales ............................................................................ 43 11 )ROGLQJRIĮ-helical membrane proteins in the membrane .................. 46 11.1 Helix-helix interactions ................................................................................ 47 11.2 van der Waals interactions .......................................................................... 47 11.3 Salt bridges ................................................................................................. 47 11.4 Hydrogen bonds .......................................................................................... 47 11.5 The GxxxG motif ......................................................................................... 48 11.6 Knobs-into-holes packing ............................................................................ 48 12 Translational arrest peptides ............................................................... 49 12.1 The biology of arrest peptides ..................................................................... 50 12.2 The ribosome exit tunnel ............................................................................. 51 12.3 Interactions between the ribosomal tunnel and passing nascent chains ..... 52 12.4 Nascent-chain induced translational stalling ................................................ 52 12.5 Features of arrest peptides ......................................................................... 53 12.5.1 The SecM arrest peptide........................................................................ 53 12.5.2 The XBP1 arrest peptide........................................................................ 54 12.5.3 Release of arrest-peptide mediated translational stalling ....................... 55 12.5.4 Other amino acid clusters can arrest translation .................................... 55 12.5.5 Laboratory-engineered arrest peptides .................................................. 56 13 Methodology ........................................................................................ 57 13.1 Leader peptidase – a model protein (Papers I-IV) ....................................... 57 13.2 In vitro protein expression (Papers I-IV) ...................................................... 58 13.3 Lep constructs (Papers I-IV) ........................................................................ 58 13.4 Determining the propensity of hydrophobic segments to integrate into the ER membrane by in vitro experiments (Papers I, II) .......................................................... 59 13.5 “Pulling force” measurements in vitro and in vivo with the use of arrest peptides (Papers III, IV) .............................................................................................. 60 13.6 Determination of the minimal glycosylation distance ................................... 61 13.7 Circular Dichroism Spectroscopy ................................................................ 61 13.8 Reversed-Phase High-Performance Liquid Chromatography (Papers I, II) . 62 14 Summary of publications ..................................................................... 63 14.1 Paper I - Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences ................................................... 63 14.2 Paper II - Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo ........................................................