Structural Genomics on Membrane Proteins

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ISBN: 9781574445268 | 157444526X
Cover: Hardcover
Copyright: 2/23/2006

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While the genomic revolution has quickly led to the deposit of more than 30,000 structures in the protein data bank (PDB), less than one percent of those contributions represent membrane proteins despite the fact that membrane proteins constitute some 20 percent of all proteins. This discrepancy becomes significantly troublesome when it is coupled with the fact that 60 percent of current drugs are based on targeting this group of proteins, a trend that does not seem likely to reverse.Structural Genomics on Membrane Proteins provides an excellent overview on novel research in bioinformatics and modeling on membranes, as well as the latest technological developments being employed in expression, purification, and crystallography to obtain high-resolution structures on membrane proteins. This cutting-edge work also explains the difficulties facing researchers-both technical and ethical-that have slowed the process.Structural Genomics on Membrane Proteins provides researchers with an unprecedented look at the novel technologies that will ultimately allow them to conquer the last frontier in structural biology, leading to accelerated breakthroughs in drug discovery.

Chapter 1 Introduction

(4)

Kenneth H. Lundstrom

1.1 Scope of Book

(2)

1.2 Summary

(1)

References

(2)

Chapter 2 Bioinformatics in Membrane Protein Analysis

(16)

Bengt Persson

2.1 Introduction

(1)

2.2 Prediction of Membrane-Spanning Regions of Proteins

(7)

2.2.1 Hydrophobicity Analysis

(2)

2.2.2 The Positive Inside Rule

(1)

2.2.3 Use of Multiple Sequence Alignments

(1)

2.2.4 Model-Recognition Approaches

(1)

2.2.5 Support Vector Machines

(1)

2.2.6 Consensus Techniques

(1)

2.2.7 Prediction of Beta-Barrel Proteins

(1)

2.3 Prediction Confidence

(1)

2.3.1 Partial Predictions with High Accuracy

(1)

2.3.2 Combination of Predictions and Experimental Determination

(1)

2.4 Evaluation of Methods

(1)

2.5 Test Sets and Databases of Membrane Proteins

(1)

2.6 Conclusion

(1)

References

(5)

Chapter 3 Prokaryotic Membrane Transport Proteins: Amplified Expression and Purification

(22)

Joanne Clough, Massoud Saidijam, Kim Bettaney, Gerda Szakonyi, Simon Patching, Johan Meuller, Shun'ichi Suzuki, Keigo Shibayama, Mark Bacon, Emma Barksby, Marie Groves, Richard Herbert, Mary Phillips-Jones, Alison Ward, Frank Gunn-Moore, John O'Reilly, Nick Rutherford, Roslyn Bill, and Peter Henderson

3.1 Introduction

(3)

3.2 E. coli Expression Systems

(2)

3.3 Optimization of Conditions for Expression

(4)

3.4 Identification of Overexpressed Membrane Proteins

(1)

3.5 Purification of Membrane Proteins Facilitated by the Addition of Affinity Tags

(2)

3.6 Choice of Detergent and Solubilization Conditions

(1)

3.7 Reconstitution and Assay of Membrane Protein Activity

(1)

3.8 Physical Measurements on Detergent-Solubilized and Reconstituted Membrane Protein

(1)

3.9 Crystallization of Purified Membrane Transport Proteins

(1)

3.10 Nuclear Magnetic Resonance (NMR) Approaches

(1)

3.11 Conclusion

(1)

Acknowledgments

(1)

References

(6)

Chapter 4 Membrane Protein Production Strategies for Structural Genomics

(14)

Said Eshaghi and Pär Nordlund

4.1 Introduction

(1)

4.2 General Aspects of Membrane Protein Production Technologies

(1)

4.3 E. coli Expression Strategies

(1)

4.4 Small-Scale Parallel Expression-Screening Strategies

(1)

4.5 Benchmarking of Expression Strategies

(3)

4.6 Parallel Detergent Screening

(2)

4.7 Future Prospects

(2)

Acknowledgments

(1)

References

(4)

Chapter 5 Refolding of Membrane Proteins for Large-Scale Production

(16)

Hans Kiefer, Thomas Ostermann, and Monika Bähner

5.1 Introduction

(2)

5.2 Theoretical Considerations

(3)

5.2.1 The Two-Stage Model of Membrane Protein Folding

(1)

5.2.2 Stability of Membrane Proteins

(1)

5.2.3 Is There a Rational Approach to Membrane Protein Refolding?

(1)

5.3 Membrane Protein Refolding in Practice

(5)

5.3.1 Bacteriorhodopsin

(1)

5.3.2 Light-Harvesting Complex

(1)

5.3.3 Diacylglycerol Kinase

(1)

5.3.4 Disulfide Bond–Reducing Protein B

(1)

5.3.5 Mitochondrial Transport Proteins

(1)

5.3.6 Potassium Channel KcsA

(1)

5.3.7 Olfactory Receptor OR5

(1)

5.3.8 Leukotriene Receptor BLT1

(1)

5.4 Strategies to Identify (Re-)Folding Conditions

(2)

5.5 Future Prospects

(1)

References

(4)

Chapter 6 Crystallization of Membrane Proteins

(16)

Alastair T. Gardiner, June Southall, and Richard J. Cogdell

6.1 Introduction

(3)

6.2 Practical Considerations

(4)

6.2.1 Detergent Choice for Sample Preparation

(1)

6.2.2 Choice of Detergents for Crystallization Trials

(1)

6.2.3 Choice of Precipitant

(1)

6.2.4 Phase Separation and the Small Amphiphile Concept

(2)

6.2.5 Choice of Temperature and pH

(1)

6.3 Crystallization Strategies

(4)

6.3.1 Vapor Diffusion

(1)

6.3.2 Microdialysis

(1)

6.3.3 Co-crystallization

(1)

6.3.4 Lipidic Cubic Phase

(1)

6.3.5 Type III Crystallization Method

(1)

6.4 Future Prospects

(1)

Acknowledgments

(1)

References

(4)

Chapter 7 Signaling through Membrane Proteins

(26)

J. Robert Bostwick and Deborah S. Hartman

7.1 Introduction

(1)

7.2 Molecular Mechanisms of Signaling

(7)

7.2.1 G Protein-Coupled Receptors

(2)

7.2.2 Ion Channels

(1)

7.2.3 Single-Membrane-Spanning Domain Receptors

(1)

7.3 Pharmacology of Signaling

(5)

7.3.1 GPCR Agonist-Induced Trafficking

(1)

7.3.2 Allosteric Modulation

100

(2)

7.4 Detection Methods for Receptor Signaling

102

(3)

7.5 The Role of Receptor Complexes in Cell Signaling

105

(1)

7.6 Conclusion

106

(1)

References

107

(8)

Chapter 8 Expression of Membrane Proteins in Yeasts

115

(38)

Christoph Reinhart and Christoph Krettler

8.1 Introduction

115

(2)

8.2 Yeast Expression Systems Used for Membrane Protein Production

117

(1)

8.2.1 Saccharomyces cerevisiae

117

(1)

8.2.2 Schizosaccharomyces pombe

117

(1)

8.2.3 Pichia pastoris

118

(1)

8.3 Production Overview

118

(17)

8.3.1 Receptors

118

(8)

8.3.1.1 Production of GPCRs in S. cerevisiae

119

(4)

8.3.1.2 Production of GPCRs in S. pombe

123

(1)

8.3.1.3 Production of GPCRs in P. pastoris

124

(2)

8.3.2 Transport Proteins

126

(9)

8.4 Functional Assays in Yeast Systems

135

(6)

8.4.1 Pharmacological Ligand Screening Assays for GPCRs

135

(2)

8.4.2 Detection of Interactions of Membrane Proteins: The Split-Ubiquitin Membrane Yeast Two-Hybrid System

137

(4)

8.5 Conclusion

141

(1)

Acknowledgments

142

(1)

References

142

(11)

Chapter 9 Expression of Functional Membrane Proteins in the Baculovirus–Insect Cell System: Challenges and Developments

153

(16)

Giel J.C.G.M. Bosman and Willem J. de Grip

9.1 Introduction

153

(2)

9.2 The Baculovirus System

155

(3)

9.2.1 Recombinant Virus

155

(1)

9.2.2 Cell Lines

156

(1)

9.2.3 Optimization

157

(1)

9.3 Glycosylation

158

(1)

9.4 Improving the Yield of Functional Proteins

159

(2)

9.5 New Developments

161

(1)

9.6 Conclusion

162

(1)

References

163

(6)

Chapter 10 Expression of Membrane Proteins in Mammalian Cells

169

(10)

Kenneth H. Lundstrom

10.1 Introduction

169

(1)

10.2 Transient Transfection

170

(2)

10.3 Stable Cell Lines

172

(1)

10.4 Adenovirus

172

(1)

10.5 Vaccinia Virus

173

(1)

10.6 Lentivirus

173

(1)

10.7 Semliki Forest Virus

174

(1)

10.8 Conclusion

175

(1)

References

175

(4)

Chapter 11 Solubilization and Purification of Membrane Proteins

179

(20)

Bernadette Byrne and Mika Jormakka

11.1 Introduction

179

(1)

11.2 Membrane Partitioning

180

(1)

11.3 Solubilization

180

(8)

11.3.1 Detergents

180

(4)

11.3.2 Types of Detergents

184

(1)

11.3.3 Solubilization Procedures

184

(3)

11.3.4 Alternative Means of Solubilizing Membrane Proteins

187

(1)

11.4 Purification

188

(5)

11.4.1 Affinity Chromatography

188

(2)

11.4.2 Disadvantages of His-tags

190

(1)

11.4.3 Other Means of Purification

191

(1)

11.4.4 Ion Exchange Chromatography

191

(1)

11.4.5 Size Exclusion

192

(1)

11.5 Aggregation of Membrane Proteins

193

(1)

11.6 SDS-PAGE Analysis of Membrane Proteins

194

(1)

11.7 Removal/Exchange of Detergent

195

(1)

11.8 Future Prospects

196

(1)

References

197

(2)

Chapter 12 Fluorescent Labelling of Membrane Proteins in Living Cells

199

(12)

Ruud Hovius, Bruno H. Meyer, Emmanuel G. Guignet, and Horst Vogel

12.1 Introduction

199

(2)

12.2 Biosynthetical Labelling

201

(2)

12.2.1 Fluorescent Proteins

201

(1)

12.2.2 Artificial Amino Acids

202

(1)

12.2.3 C-Terminal Labelling

202

(1)

12.3 Postbiosynthetic Labelling

203

(4)

12.3.1 Fusion Proteins with Enzymatic Activity

203

(1)

12.3.2 Protein Fusions as Substrates

204

(1)

12.3.3 Fusion Proteins with Binding Activity

204

(1)

12.3.4 Recognition Tags

204

(3)

12.4 Conclusion

207

(1)

References

207

(4)

Chapter 13 Membrane Protein NMR

211

(50)

Xiang-Qun (Sean) Xie

13.1 Introduction

211

(2)

13.2 Production of Membrane Proteins for NMR Studies

213

(7)

13.2.1 Overview of Membrane Protein Expression Systems

214

(1)

13.2.2 Overview of Membrane Protein Purification

215

(1)

13.2.3 Membrane Protein Solubilization and Renaturation

216

(4)

13.2.3.1 Quasi-Native Membrane Environment

218

(1)

13.2.3.2 Native-Like Membrane Environment

218

(2)

13.3 High-Resolution Solution-State NMR for Transmembrane Proteins

220

(11)

13.3.1 Sequential Assignment

221

(4)

13.3.2 Conformational Data Measurements

225

(6)

13.3.2.1 Chemical Shift Index

225

(2)

13.3.2.2 NOE Distance Constraints

227

(1)

13.3.2.3 Torsional Angle Constraints

227

(1)

13.3.2.4 Hydrogen-Bonding Constraints

228

(1)

13.3.2.5 Residual Dipolar Coupling Constraints

229

(2)

13.4 High-Resolution Solid-State NMR for Transmembrane Proteins

231

(3)

13.4.1 Magic Angle Sample Spinning NMR

232

(1)

13.4.2 Uniaxially Aligned Samples NMR

233

(1)

13.5 Membrane Protein NMR Strategies and Developments

234

(7)

13.5.1 Truncated Membrane Protein Fragments — Cannabinoid CB2 Receptor

235

(2)

13.5.2 Two-Transmembrane-Helices Protein — Mercury Transport Protein MerF

237

(2)

13.5.3 Three-Helix-Bundle Transmembrane Protein — DAGK

239

(1)

13.5.4 Four-Helix-Bundle Transmembrane Protein— MISTIC

240

(1)

Acknowledgments

241

(1)

References

242

(19)

Chapter 14 Miniaturization of Structural Biology Technologies — From Expression to Biophysical Analyses

261

(38)

Enrique Abola, Peter Kuhn, and Raymond C. Stevens

14.1 Introduction

261

(1)

14.2 New Tools for High-Throughput Structure Determination Pipelines

262

(17)

14.2.1 Microexpression Protocols

263

(2)

14.2.2 Microcoil NMR Probe

265

(1)

14.2.3 Nanocalorimeter or "Enthalpy Array"

266

(3)

14.2.4 Automated Electron Microscopy and Table-Top Electron Microscopy Screening

269

(3)

14.2.5 Miniaturization and Automation of the Crystallization Experiment

272

(7)

14.2.5.1 Integrated Nanovolume Crystallization System

276

(3)

14.3 Results from Five Years of Nanovolume Crystallization Experiments

279

(13)

14.3.1 Exploring Chemical Space

279

(7)

14.3.2 Biophysical Properties of Proteins in Thermotoga Maritima That Crystallized

286

(3)

14.3.3 Nanovolume Crystallization and Crystal Quality

289

(3)

14.4 Future Directions

292

(1)

Acknowledgments

293

(1)

References

293

(6)

Chapter 15 Electron Microscopy and Atomic Force Microscopy of Reconstituted Membrane Proteins

299

(22)

Andreas Engel

15.1 Introduction

300

(1)

15.2 Two-Dimensional Crystallization

301

(3)

15.2.1 Different Methods for 2D Crystallization

301

(1)

15.2.2 Critical Parameters

302

(2)

15.3 Electron Microscopy

304

(7)

15.3.1 Image Formation

304

(2)

15.3.2 Electron Diffraction

306

(1)

15.3.3 Specimen Preparation

307

(1)

15.3.4 Data Processing

307

(4)

15.4 Atomic Force Microscopy

311

(6)

15.4.1 Image Formation

311

(1)

15.4.2 Observing Membrane Proteins at Work

312

(1)

15.4.3 Imaging Native Membranes

312

(3)

15.4.4 Nanomanipulation

315

(1)

15.4.5 Data Processing

316

(1)

15.5 Conclusion

317

(1)

Acknowledgments

317

(1)

References

317

(4)

Chapter 16 Structural Genomics Networks for Membrane Proteins

321

(10)

Kenneth H. Lundstrom

16.1 Introduction

321

(1)

16.2 Networks on Prokaryotic Targets

322

(2)

16.3 Networks on Prokaryotic and Eukaryotic Targets

324

(1)

16.4 Networks on Eukaryotic Targets

325

(2)

16.5 MePNet

327

(1)

16.6 Conclusion

328

(1)

References

328

(3)

Chapter 17 Molecular Modelling of Membrane Proteins

331

(18)

Slawomir Filipek and Anna Modzelewska

17.1 Introduction

331

(1)

17.2 Protein Structure Prediction

332

(1)

17.3 Evaluation of Predicted Models

333

(1)

17.4 Structure Optimization and Analysis

334

(2)

17.5 Modelling Protein—Ligand Complexes

336

(2)

17.6 Modelling Protein—Protein Complexes

338

(1)

17.7 Application to Complex of Rhodopsin and Rhodopsin Kinase

339

(5)

17.7.1 Molecular Model of the Rhodopsin Oligomer

339

(1)

17.7.2 Modelling Rhodopsin Kinase and Its Complex with Rhodopsin Oligomer

340

(4)

17.8 Future Prospects

344

(1)

References

344

(5)

Chapter 18 Toward Structural Bases for GPCR Ligand Binding: A Path for Drug Discovery

349

(30)

Sabrina A. Beretta, Alla Korepanova, Mulugeta Mamo, Geoffrey E. Stamper, and Mark L. Chiu

18.1 Introduction

349

(3)

18.2 Drug Discovery via GPCR Ligand Binding and Functional Assay Screening

352

(1)

18.3 The Value of Structure-Based Drug Design of GPCRs

353

(1)

18.4 Localization of GPCR Ligand-Binding Domains

354

(6)

18.4.1 Ligand-Binding Domains of Family 1 GPCRs

355

(2)

18.4.2 Ligand-Binding Domains of Family 2 GPCRs

357

(1)

18.4.3 Ligand-Binding Domains of Family 3 GPCRs

358

(2)

18.5 NMR of GPCRs

360

(3)

18.5.1 Solution NMR Studies of Ligand Interactions with GPCRs

360

(1)

18.5.2 Solid-State NMR Studies of Ligand Interactions with GPCRs

361

(2)

18.6 Molecular Modelling of GPCRs from the Rhodopsin Structure

363

(2)

18.7 Conclusion

365

(2)

References

367

(12)

Index

379

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Structural Genomics on Membrane Proteins

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