Preface ....................................................... VII
List of Contributors .......................................... XIX
Part 1 How Lipids Shape Proteins
1 Lipid Bilayers, Translocons and the Shaping of
Polypeptide Structure ...................................... 3
Stephen H. White, Tara Hessa, and Gunnar von Heijne
1.1 Introduction ............................................... 3
1.2 Membrane Proteins: Intrinsic Interactions .................. 5
1.2.1 Physical Determinants of Membrane Protein
Stability: The Bilayer Milieu ....................... 5
1.2.2 Physical Determinants of Membrane Protein
Stability: Energetics of Peptides in Bilayers ....... 9
1.2.3 Physical Determinants of Membrane Protein
Stability: Helix-Helix Interactions in Bilayers .... 13
1.3 Membrane Proteins: Formative Interactions ................. 14
1.3.1 Connecting Translocon-assisted Folding to
Physical Hydrophobicity Scales: The Interfacial
Connection ......................................... 14
1.3.2 Connecting Translocon-assisted Folding to
Physical Hydrophobicity Scales: Transmembrane
Insertion of Helices ............................... 16
1.4 Perspectives .............................................. 21
References ................................................ 22
2 Folding and Stability of Monomeric ^-Barrel Membrane
Proteins .................................................. 27
Jörg H. Kleinschmidt
2.1 Introduction .............................................. 27
2.2 Stability of β-Barrel Membrane Proteins ................... 29
2.2.1 Thermodynamic Stability of FepA in Detergent
Micelles ........................................... 29
2.2.2 Thermodynamic Stability of OmpA in Phospholipids
Bilayers ........................................... 30
2.2.3 Thermal Stability of FhuA in Detergent Micelles .... 31
2.3 Insertion and Folding of Transmembrane β-Barrel
Proteins .................................................. 32
2.3.1 Insertion and Folding of β-Barrel Membrane
Proteins in Micelles ............................... 32
2.3.2 Oriented Insertion and Folding into Phospholipid
Bilayers ........................................... 32
2.3.3 Assemblies of Amphiphiles Induce Structure
Formation in β-Barrel Membrane Proteins ............ 33
2.3.4 Electrophoresis as a Tool to Monitor Insertion
and Folding of β-Barrel Membrane Proteins .......... 34
2.3.5 pH and Lipid Headgroup Dependence of the Folding
of β-Barrel Membrane Proteins ...................... 35
2.4 Kinetics of Membrane Protein Folding ...................... 35
2.4.1 Rate Law for β-Barrel Membrane Protein Folding
and Lipid Acyl Chain Length Dependence ............. 35
2.4.2 Synchronized Kinetics of Secondary and Tertiary
Structure Formation of the β-Barrel OmpA ........... 36
2.4.3 Interaction of OmpA with the Lipid Bilayer is
Faster than the Formation of Folded OmpA ........... 36
2.5 Folding Mechanism of the β-Barrel of OmpA
into DOPC Bilayers ........................................ 37
2.5.1 Multistep Folding Kinetics and Temperature
Dependence of OmpA Folding ......................... 37
2.5.2 Characterization of Folding Intermediates by
Fluorescence Quenching ............................. 38
2.5.3 The β-Barrel Domain of OmpA Folds and Inserts by
a Concerted Mechanism .............................. 40
2.6 Protein-Lipid Interactions at the Interface of
β-Barrel Membrane Proteins ................................ 42
2.6.1 Stoichiometry of the Lipid-Protein Interface ....... 42
2.6.2 Lipid Selectivity of β-Barrel Membrane Proteins .... 42
2.7 Orientation of β-Barrel Membrane Proteins in Lipid
Bilayers .................................................. 43
2.7.1 Lipid Dependence of the β-Barrel Orientation
Relative to the Membrane ........................... 43
2.7.2 Inclination of the β-Strands Relative to the
β-Barrel Axis in Lipid Bilayers .................... 44
2.7.3 Hydrophobic Matching of the β-Barrel and the
Lipid Bilayer ...................................... 44
2.8 In vivo Requirements for the Folding of OMPs .............. 45
2.8.1 Amino Acid Sequence Constraints for OmpA Folding
in vivo ............................................ 45
2.8.2 Periplasmic Chaperones ............................. 45
2.8.3 Insertion and Folding of the β-Barrel OmpA is
Assisted by Skp and LPS ............................ 46
2.8.4 Role of Omp85 in Targeting or Assembly of
β-Barrel Membrane Proteins ......................... 48
2.9 Outlook ................................................... 51
References ................................................ 52
3 A Paradigm of Membrane Protein Folding: Principles,
Kinetics and Stability of Bacteriorhodopsin Folding ....... 57
Paula J. Booth
3.1 Introduction .............................................. 57
3.2 Principles of Transmembrane α-Helical Membrane Protein
Folding: A Thermodynamic Model for Bacteriorhodopsin ...... 59
3.3 Bacteriorhodopsin Stability ............................... 60
3.3.1 Side-chain Contributions to Helix Interactions
and the Role of Pro ................................ 61
3.3.2 Helix-connecting Loops ............................. 62
3.4 Pulling the Protein Out of the Membrane ................... 63
3.5 Bacteriorhodopsin Folding Kinetics ........................ 64
3.5.1 Cotranslational Insertion .......................... 65
3.5.2 Retinal Binding Studies to Apomembrane ............. 65
3.5.3 Unfolding, Refolding and Kinetic Studies in vitro .. 67
3.6 Controlling Membrane Protein Folding ...................... 69
3.7 Conclusions ............................................... 71
3.7.1 Summary of Bacteriorhodopsin Folding ............... 71
3.7.2 Implications for Transmembrane a-Helical Membrane
Protein Folding .................................... 73
References ................................................ 75
4 Post-integration Misassembly of Membrane Proteins and
Disease ................................................... 81
Charles R. Sanders
4.1 Introduction .............................................. 81
4.2 A Given IMP May be Subject to Numerous Disease-linked
Mutations ................................................. 82
4.3 Ligand Rescue of Misassembly-prone Membrane Proteins:
Implications .............................................. 84
4.4 What IMP Properties Affect Folding Efficiency in the
Cell? ..................................................... 87
4.5 Common Mutations in Transmembrane Domains That Lead to
Misassembly and Disease ................................... 89
4.6 Correlating Biophysical, Cell-biological and Biomedical
Measurements .............................................. 90
References ................................................ 91
Part 2 How Proteins Shape Lipids
5 A Census of Ordered Lipids and Detergents in X-ray
Crystal Structures of Integral Membrane Proteins .......... 97
Michael C. Wiener
5.1 Introduction .............................................. 97
5.2 Results ................................................... 98
5.3 Illustrative Examples of Selected Bound Lipids,
Detergents and Related Molecules ......................... 103
5.3.1 Integral Membrane Protein Structures Contain
Ordered Native Lipids ............................. 103
5.3.2 Structures of Lipids in Membrane Protein
Co-crystals Differ from Those in Pure Lipid
Crystals .......................................... 107
5.3.3 Native Lipids can Stabilize and Preserve
Protein-Protein Interfaces ........................ 108
5.3.4 Multiple Acyl Chain Conformations Exist for
Efficient Packing with Protein Interfaces ......... 108
5.3.5 Lipid Acyl Chains Interact Primarily with
Aliphatic and Aromatic Amino Acid Side-chains ..... 109
5.3.6 Unusual Lipid/Detergent Conformations Occur at
the Protein-Lipid Interface ....................... 109
5.3.7 A Bilayer Structure is Present in Crystals Grown
from the LCP ...................................... 112
5.4 Conclusion ............................................... 114
References ............................................... 115
6 Lipid and Detergent Interactions with Membrane Proteins
Derived from Solution Nuclear Magnetic Resonance ......... 119
Ashish Arora
6.1 Introduction ............................................. 119
6.2 Heteronuclear Solution NMR of Protein/Detergent
Complexes ................................................ 120
6.3 Choice of Detergents ..................................... 122
6.4 Size and Shape of Pure Detergent Micelles and
Detergent/Protein Complexes .............................. 124
6.5 Sample Preparation for Solution NMR Measurements ......... 125
6.6 Protein/Detergent Interactions Monitored by NMR
Spectroscopy ............................................. 228
6.7 Dynamics and Conformational Transitions of Membrane
Proteins in Detergent Micelles ........................... 130
6.8 MD Simulations of Protein/Detergent Complexes ............ 131
6.9 Implications on the Structure and Function of Membrane
Proteins in Biological Membranes ......................... 133
References ............................................... 134
Part 3 Membrane Penetration by Toxins
7 Lipid Interactions of a-Helical Protein Toxins ........... 141
Gregor Anderluh and Jeremy H. Lakey
7.1 Introduction ............................................. 141
7.1.1 The Two Secondary Structures Compared ............. 141
7.1.2 Lessons from a Potassium Channel .................. 145
7.2 Pore-forming Colicins .................................... 145
7.2.1 Outer Membrane Interactions ....................... 146
7.2.2 Colicin A Requires Acidic Lipids .................. 147
7.2.3 The Open Channel .................................. 148
7.2.4 The Colicin-Phospholipid Complex .................. 149
7.2.5 Other Similar Proteins ............................ 150
7.3 Actinoporins ............................................. 151
7.3.1 Initial Lipid Binding ............................. 152
7.3.2 Helix Insertion ................................... 154
7.3.3 The Oligomeric Pore ............................... 155
7.4 Conclusion ............................................... 156
References ............................................... 157
8 Membrane Recognition and Pore Formation by Bacterial
Pore-forming Toxins ...................................... 163
Alejandro P. Heuck and Arthur E. Johnson
8.1 Introduction ............................................. 163
8.2 Classification of Bacterial PFTs ......................... 163
8.2.1 α-PFTs ............................................ 164
8.2.2 β-PFTs ............................................ 166
8.3 A General Mechanism of Pore Formation? ................... 166
8.4 Membrane Recognition ..................................... 169
8.4.1 Recognition of Specific Membrane Lipids ........... 170
8.4.2 Recognition of Membrane-anchored Proteins or
Carbohydrates ..................................... 172
8.4.3 The Role of Membrane Lipid Domains ................ 173
8.5 Oligomerization on the Membrane Surface .................. 175
8.5.1 Oligomerization Triggered by Lipid-induced
Conformational Changes ............................ 176
8.5.2 Oligomerization Following Proteolytic Activation
of Toxins ......................................... 178
8.6 Membrane Penetration and Pore Formation .................. 179
8.7 Unresolved Issues ........................................ 181
References ............................................... 183
9 Mechanism of Membrane Permeation and Pore Formation by
Antimicrobial Peptides ................................... 187
Yechiel Shai
9.1 Introduction ............................................. 187
9.2 The Cell Membrane is the Major Binding Site for Most
Cationic Antimicrobial Peptides .......................... 188
9.2.1 Non-receptor-mediated Interaction of
Antimicrobial Peptides with their Target Cells .... 189
9.2.2 A Receptor-mediated Interaction of Antimicrobial
Peptides with their Target Cells .................. 191
9.3 Parameters Involved in the Selection of Target Cells by
Antimicrobial Peptides ................................... 192
9.3.1 The Role of the Composition of the Cell Wall and
the Cytoplasmic Membrane .......................... 193
9.3.2 The Role of the Peptide Chain and Its
Organization ...................................... 194
9.4 The Lethal Event Caused by Antimicrobial Peptides ........ 201
9.5 How do Antimicrobial Peptides Damage the Integrity of
the Target Cell Membrane? ................................ 202
9.5.1 Membrane-imposed Amphipathic Structure ............ 202
9.5.2 Molecular Mechanisms of Membrane Permeation ....... 204
9.5.3 The Molecular Architecture of the Permeation
Pathway ........................................... 208
9.6 Summary and Conclusions .................................. 209
References ............................................... 210
Part 4 Mechanisms of Membrane Fusion
10 Cell Fusion in Development and Disease ................... 221
Benjamin Podbileivicz and Leonid V. Chernomordik
10.1 Introduction ............................................. 221
10.2 Developmental Cell Fusion for Health ..................... 221
10.2.1 Muscles ........................................... 222
10.2.3 Comparison between Cell Fusion in a Worm, a
Fly and Vertebrates ............................... 231
10.3 Cell Fusion in Diseases .................................. 233
10.3.1 Cell Fusion Mediated by Enveloped Viruses ........ 233
10.4 Dissection of Developmental Fusion Based on Viral
Fusion Analogies ......................................... 239
10.4.1 Activation of a Developmental Fusogen ............. 239
10.4.2 Dissection of Developmental Cell Fusion ........... 239
10.4.3 Direct Cell Fusion Promotion or Indirect
Relaxation of Fusion Blocks ....................... 240
10.5 Concluding Remarks ....................................... 240
References ............................................... 241
11 Molecular Mechanisms of Intracellular Membrane Fusion .... 245
Olga Vites and Reinhard Jahn
11.1 Introduction ............................................. 245
11.2 Intracellular Fusion Reactions - An Overview ............. 246
11.3 Tethering and Docking .................................... 247
11.4 SNARE Proteins - The Fusion Catalysts? ................... 249
11.4.1 Assembly-Disassembly Cycle of SNARE Proteins ...... 249
11.4.2 N-terminal Domains of SNAREs - Recruiting
Proteins or Regulating SNARE Function? ............ 251
11.4.3 "Zippering" Model for SNARE-mediated Membrane
Fusion ............................................ 252
11.4.4 Trans-complexes - Intermediates in the Fusion
Pathway? .......................................... 253
11.4.5 Acceptor Complexes, Topology and Specificity ...... 256
11.5 SM Proteins and Other Regulators ......................... 262
11.5.1 SM Proteins ....................................... 263
11.6 Fusion Pores ............................................. 264
11.6.1 Measuring Fusion Pore Opening and Closure ......... 265
11.6.2 The Role of Proteins in Controlling Fusion
Pore Opening ...................................... 266
11.7 Concluding Remarks ....................................... 267
List of Abbreviations .................................... 267
References ............................................... 268
12 Interplay of Proteins and Lipids in Virus Entry by
Membrane Fusion .......................................... 279
Alex L. Lai, Yinling Li, and Lukas К. Tamm
12.1 Introduction ............................................. 279
12.2 Fusion of Pure Lipid Bilayers ............................ 281
12.3 Viral Protein Sequences that Mediate Lipid Bilayer
Fusion ................................................... 284
12.3.1 Fusion Peptides ................................... 284
12.3.2 Transmembrane Domains ............................. 285
12.3.3 Other Regions of the Fusion Protein ............... 285
12.4 Interactions of Fusion Peptides with Lipid Bilayers ...... 286
12.4.1 HIV Fusion Peptide-Bilayer Interactions ........... 287
12.4.2 Influenza Fusion Peptide Structure ................ 288
12.4.3 Influenza Fusion Peptide Mutants .................. 290
12.4.4 Binding of Fusion Peptides to Lipid Bilayers ...... 290
12.4.5 Sendai, Measles and Ebola Fusion Peptide-Bilayer
Interactions ...................................... 290
12.4.6 Perturbation of Bilayer Structure by Fusion
Peptides .......................................... 291
12.5 Interactions of Transmembrane Domains with Lipid
Bilayers ................................................. 292
12.6 Structure-Function (Fusion) Relationships of Membrane-
interactive Viral Fusion Protein Domains ................. 294
12.6.1 Fusion Peptide Mutants ............................ 294
12.6.2 Transmembrane Domain Mutants ...................... 295
12.7 Possible Mechanisms for Initiating the Formation of
Viral Fusion Pores ....................................... 296
References ............................................... 300
Part 5 Cholesterol, Lipid Rafts, and Protein Sorting
13 Protein-Lipid Interactions in the Formation of Raft
Microdomains in Biological Membranes ..................... 307
Akihiro Kusumi, Kenichi Suzuki, Junko Kondo, Nobuhiro
Morone, and Yasuhiro Umemura
13.1 Many Plasma Membrane Functions are Mediated by Molecular
Complexes, Microdomains and Membrane Skeleton-based
Compartments ............................................. 307
13.2 Timescales, Please! ...................................... 309
13.3 Four Types of Membrane Domains ........................... 310
13.4 The Cell Membrane is a Two-dimensional Non-ideal Liquid
Containing Dynamic Structures on Various Time-Space
Scales ................................................... 324
13.5 A Definition of Raft Domains ............................. 325
13.6 The Original Raft Hypothesis ............................. 316
13.7 Are there Raft Domains in Steady-state Cells in the
Absence of Extracellular Stimulation? .................... 316
13.7.1 Standard Immunofluorescence or Immunoelectron
Microscopy Failed to Detect Raft-like Domains
in the Plasma Membrane of Steady-state Cells ...... 317
13.7.2 The Recovery of a Molecule in Detergent-
resistant Membrane (DRM) Fractions Might Infer
its Raft Association in the Cell Membrane, but
the Relationship between DRM Fractions and Raft
Domains is Complicated ............................ 317
13.7.3 The Size of Rafts in Plasma Membranes of Steady-
state Cells may be 10 nm or Less .................. 319
13.7.4 Mushroom Model for the Steady-state Raft .......... 322
13.8 Stabilized Rafts Induced by Protein Clustering in Plasma
and Golgi Membranes ...................................... 324
13.8.1 Clustering of Raft Molecules by Ligand Binding
or Crosslinking Induces Stabilized Rafts
("Receptor-cluster Rafts") ........................ 324
13.8.2 How can Raft Molecule Clustering Induce
Stabilized Rafts? ................................. 324
13.9 Can Receptor-cluster Rafts Work as Platforms to
Facilitate the Assembly of Raftophilic Molecules? ........ 326
13.9.1 Benchmarks for Experiments Examining the
Colocalization of Raftophilic Molecules ........... 326
13.9.2 Simultaneous Crosslinking of Two GPI-anchored
Receptors ......................................... 327
13.9.3 Sequential Crosslinking of One Species of
GPI-anchored Receptors Followed by Crosslinking
of a Second Species without Fixation .............. 328
13.9.4 Examination of the Recruitment of
Non-crosslinked Second Raftophilic Molecules to
Crosslinked GPI-anchored Receptor Clusters ........ 328
13.9.5 Difficulty in Colocalization Experiments using
Raftophilic Molecules: Low Levels of
Colocalization and Quantitative Reproducibility
Due to Sensitivity to Subtle Differences in
Experimental Conditions and Protocols ............. 329
13.10 Timescales Again! Transient Colocalization of
Raftophilic Molecules ................................... 329
13.11 Modified Raft Hypothesis ................................ 331
References .............................................. 332
14 Protein and Lipid Partitioning in Locally Heterogeneous
Model Membranes .......................................... 337
Petra Schwille, Nicoktta Kahya, and Kirsten Bacia
14.1 Introduction: Why Should We Use Simple Model Membranes
to Gain Insight into Complex Membrane Organization? ...... 337
14.1.1 Relation of Domain Structure to a Biological
Function .......................................... 337
14.1.2 An Accessible Detection Method .................... 338
14.1.3 The Term "Raff .................................... 338
14.2 Biomimetic Membranes ..................................... 340
14.2.1 GUVs: Properties and Preparation .................. 342
14.3 Methods of Investigation of Domain Formation in
Biomimetic Membranes ..................................... 343
14.3.1 Electron Microscopy ............................... 343
14.3.2 Atomic Force Microscopy (AFM) ..................... 343
14.3.3 Near-field Scanning Optical Microscopy (NSOM) ..... 344
14.3.4 Fluorescence Imaging (Confocal, Multi-photon) ..... 344
14.3.5 Fluorescence Photobleaching Recovery (FPR) or
Fluorescence Recovery after Photobleaching
(FRAP) ............................................ 344
14.3.6 Single Particle Tracking (SPT) .................... 344
14.3.7 Fluorescence Correlation Spectroscopy (FCS) ....... 345
14.4 Lipid Domain Assembly in GUVs ............................ 345
14.4.1 Phase Separation .................................. 345
14.4.2 Binary Lipid Systems .............................. 348
14.4.3 Ternary Lipid Systems ............................. 351
14.4.4 Effect of Sterols on Lipid Segregation ............ 353
14.4.5 Lipid Dynamics in Domain-exhibiting GUVs .......... 354
14.5 Spatial Organization and Dynamics of Membrane
Proteins in GUVs ......................................... 357
14.6 From Model to Cellular Membranes ......................... 358
14.6.1 Model Membranes Constitute Test Systems for
Developing New and Improving Existing Detection
Techniques ........................................ 358
14.6.2 Direct Comparison Between Results Obtained on
Model and Native Membranes ........................ 361
14.6.3 Model Membranes Demonstrate What Structures Can
be Potentially Formed by Lipids and Proteins,
and Suggest Mechanisms for Fulfilling in vivo
Functions ......................................... 361
References ............................................... 362
Part 6 Targeting of Extrinsic Membrane Protein Modules to
Membranes and Signal Transduction
15 In vitro and Cellular Membrane-binding Mechanisms
of Membrane-targeting Domains ............................ 369
Wonhwa Cho and Robert V. Stahelin
15.1 Introduction ............................................. 369
15.2 Membrane Interactions of Membrane-targeting Domains ...... 370
15.2.1 Interfacial Location of Membrane-targeting
Domains ........................................... 370
15.2.2 Energetics and Kinetics of Membrane-Protein
Interactions ...................................... 371
15.3 CI Domains ............................................... 373
15.3.1 Occurrence and Structure .......................... 373
15.3.2 Lipid Specificity ................................. 374
15.3.3 Membrane-binding Mechanisms ....................... 374
15.3.4 Subcellular Localization .......................... 375
15.4 C2 Domains ............................................... 376
15.4.1 Occurrence and Structure .......................... 376
15.4.2 Lipid Specificity ................................. 376
15.4.3 Membrane Binding Mechanisms ....................... 377
15.4.4 Subcellular Localization .......................... 378
15.5 PH Domains ............................................... 378
15.5.1 Occurrence, Structure and Lipid Specificity ....... 378
15.5.2 Membrane-binding Mechanisms ....................... 380
15.5.3 Subcellular Localization .......................... 380
15.6 FYVE Domains ............................................. 380
15.6.1 Occurrence, Structure and Lipid Specificity ....... 380
15.6.2 Membrane-binding Mechanism ........................ 382
15.6.3 Subcellular Localization .......................... 383
15.7 PX Domains ............................................... 384
15.7.1 Occurrence, Structure and Lipid Specificity ....... 384
15.7.2 Membrane-binding Mechanism ........................ 385
15.7.3 Subcellular Localization .......................... 385
15.8 ENTH and ANTH Domains .................................... 387
15.8.1 Occurrence, Structure and Lipid Specificity ....... 387
15.8.2 Membrane-binding Mechanism ........................ 387
15.9 BAR Domains .............................................. 389
15.10 FERM Domains ............................................ 390
15.11 Tubby Domains ........................................... 391
15.12 Other Phosphoinositide-binding Domains .................. 391
15.13 Perspectives ............................................ 392
References ............................................... 393
16 Structure and Interactions of C2 Domains at Membrane
Surfaces ................................................. 403
David S. Cafiso
16.1 Introduction ............................................. 403
16.2 C2 Domains: Ca2+-dependent and Ca2+-independent
Membrane Binding ......................................... 404
16.3 What Drives Membrane Targeting of C2 Domains? ............ 405
16.4 Electrostatic Binding of Simple Linear Protein Motifs .... 406
16.5 The Results of Electrostatic Calculations on C2 Domains .. 408
16.6 Determining the Interactions and Positions of C2
Domains .................................................. 410
16.6.1 Site-directed Mutagenesis ......................... 410
16.6.2 Chemical Labeling ................................. 410
16.6.3 Fluorescence ...................................... 411
16.6.4 Site-directed Spin Labeling (SDSL) to Determine
C2 Domain Orientation ............................. 411
16.7 Proteins with Multiple C2 Domains ........................ 416
16.8 Interactions of Phosphoinositides with C2 Domains ........ 417
References ............................................... 418
17 Structural Mechanisms of Allosteric Regulation
by Membrane-binding Domains .............................. 423
Bertram Canagarajah, William J. Smith, and James H.
Hurley
17.1 Introduction ............................................. 423
17.2 How Membranes and PH Domains Regulate Rho Family-
specific Guanine Nucleotide Exchange Factors (GEFs) ...... 424
17.2.1 DH and PH Domain Rho GEFs ......................... 425
17.2.2 Regulation of GEF Activity by PH Domains .......... 425
17.3 Regulation of G-protein Receptor Kinase (GRK) 2 Activity
by Lipids and the Gβγ Subunit at the Membrane ............ 429
17.4 Lipid Activation of Rac-GAP Activity: /Й-Chimaerin ....... 432
17.4.1 The CI Domain of β2-Chimaerin is Buried ........... 432
17.4.2 Mechanism of Allosteric Rac-GTPase Activation by
the CI Domain ..................................... 434
References ............................................... 435
Subject Index ................................................. 437
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