METHODS AND RESULTS IN CRYSTALLIZATION OF MEMBRANE PROTEINS edited by SO IWATA
Contents Preface xiii List of Contributors xv Part I. INTRODUCTION 1 1. How to Use This Book 3 So Iwata and Bernadette Byrne 1.1. Solubilization and Purification of Membrane Proteins 5 1.2. Crystallization of Membrane Proteins 7 1.3. Detergent Selection 8 1.4. Antibody Approach 9 1.5. Where Do We Start? 9 1.6. Conclusions 10 Part II. PRINCIPLES AND TECHNIQUES IN MEMBRANE PROTEIN CRYSTALLIZATION 13 2. Solubilizing Detergents for Membrane Proteins 15 Melvin H. Keyes, Don N. Gray, Ken E. Kreh, and Charles R. Sanders 2.1. Introduction/Background 17 2.2. Need for Detergents 18 2.3. Requirement for Many and Varied Detergents 19 2.3.1. Different for Each Membrane Protein 19 2.3.2. Sugar-Based Detergents 19 2.3.3. Lipid-Like Detergents 24 2.3.4. Cyclohexyl Alkyl Detergents 26 2.3.5. Polyoxyethylene Detergents 27 2.4. Biochemical Testing of CYFOS™, FOS-MEA™, and FOS-CHOLINE® Detergents 27 2.4.1. Lipid Solubilization 27 2.4.2. Reconstitutive Refolding of a Misfolded Membrane Protein Facilitated by FOS-CHOLINE®, CYFOS™, and FOS-MEA™ Detergents 28 2.5. Strategies and Criteria for Detergent Selection 30 2.5.1. Extraction 30 2.5.2. Crystallization and Molecular Studies 31 2.5.3. Practical Observation Regarding Detergent Solubility and Purity 31 2.5.4. Price versus Risk 33 3. Crystallization of Membrane Proteins in Lipidic Cubic Phases 39 Ehud M. Landau 3.1. Introduction 41 3.2. Crystallization in Lipidic Cubic Phases 42 3.2.1. Preparing Cubic Phases 42 3.2.2. Preparing Cubic Phases for Crystallization of Membrane Proteins 45 3.2.3. Crystal Handling 50 3.3. Summary 50 4. Practical Aspects of Membrane Protein Crystallization in Lipidic Cubic Phases 57 Peter Nollert 4.1. Operation Principle and General Considerations 59 4.2. Preparation of LCP Crystallization Setups 60 4.2.1. Glass Tubes 60 4.2.2. Preparing the LCP in a Glass Tube 61 4.2.3. Mixing and Dispensing Using Syringes 61 4.2.4. Variable Parameters in LCP Crystallization Screens 63 4.3. Inspection of Crystals Grown in LCP 65 4.3.1. General Remarks 65 4.3.2. Instrumentation for Inspection 66 4.3.3. Colored Protein Crystals 67 4.3.4. Non-Colored Protein Crystals 68 4.3.5. Potential Problem Areas, False Positives 69 4.3.6. Guidelines for Observing LCP Crystallization Setups 70 5. Antibody Fragment-Mediated Crystallization of Integral Membrane Proteins: A Review 73 Christian Ostermeier 5.1. Introduction 75 5.2. Membrane Proteins 76 5.3. How can Membrane Proteins Form Well-Ordered Type II Crystals? 80 5.4. The Critical Role of the Polar Surface 81 5.5. Enlargement of the Polar Surface Area 81 5.6. Antibody Fragments 82 5.7. Production of FV Fragments 83 5.7.1. Cloning 83 5.7.2. Expression 83 5.8. FV Fragments as a Tool for Isolation and Crystallization 84 5.8.1. FV Fragments as a Tool for Purification 84 5.8.2. Conformational Epitopes 84 5.8.3. FV Fragments as a Tool for Crystallization: They Really Work! 85 5.9. Alternatives to FV Fragments 86 6. Generating Antibody Fragments for Structural Studies: A Guide 89 Bernadette Byrne and So Iwata 6.1. Introduction 91 6.2. Hybridoma Cell Line Production 92 6.3. Procedure 93 6.3.1. Immunization 93 6.3.2. Fusion 93 6.3.3. Screening of Hybridoma Cell Lines 94 6.4. Fab Fragments 95 6.5. FV Fragments 97 6.6. Summary 98 7. Crystallization of Bacterial Outer Membrane Proteins from Detergent Solutions: Porin as a Model 101 Gabriele Rummel and Jurg P. Rosenbusch 7.1. Stating the Problems 103 7.2. Considering Potential Remedies 104 7.3. Porin as a Model 106 7.4. Criteria for Rational Detergent Selection 106 7.5. Polymorphism of Detergents and Membrane Proteins in Solution: The Significance of the Phase Diagram 114 7.6. Crystallization of Porin and Other Bacterial Outer Membrane Proteins: A Brief Survey 118 7.7. Monodispersity of Building Blocks versus Micelle Dynamics: Lessons from Protein-Detergent Contacts in Crystals 118 7.8. Porin as a Paradigm: Is It a Valid Model? 121 7.9. Conclusions and Perspectives 123 8. Crystallization of Membrane Proteins in Oils 131 Naomi E. Chayen 8.1. Introduction 133 8.2. The Automated Microbatch Technique 134 8.3. Examples of Membrane Proteins Crystallized under Oil 134 8.4. Advantages of Crystallization in Oils 135 8.5. Harvesting the Crystals 136 8.6. Summary and Future Developments 137 8.7. Exercises 137 8.7.1. Materials Required 137 8.7.2. Screening Procedure 138 8.7.3. Optimization 138 PART III. EXAMPLES OF SUCCESSFUL CRYSTALLIZATION OF MEMBRANE PROTEINS 141 A. Complexes in Photosynthesis 143 9. Crystallization of Photosystem I 145 Petra Fromme 9.1. Introduction 147 9.2. Results and Discussion 148 9.3. Biological and Biochemical Parameters 149 9.3.1. The Organism 149 9.3.2. The Physiological Status of the Organism 150 9.3.3. The Quaternary Structure and the Subunit Composition 151 9.3.4. Proteolytic Digestion and Ageing of the Protein 153 9.3.5. Binding of “Ligands” 153 9.4. Physical and Chemical Parameters 155 9.4.1. Ionic Strength 155 9.4.2. Nature of Salts 159 9.4.3. pH 160 9.4.4. Temperature 161 9.4.5. Detergent 162 9.4.6. Crystallization Agents 166 9.4.7. Diffusion and Convection (Gravity/Microgravity) 167 9.5. Nucleation and Seeding Techniques—Micro- and Macroseeding 168 B. Respiratory Complexes 175 10. Crystallization of Wolinella succinogenes Quinol:Fumarate Reductase in Three Crystal Forms 177 C. Roy D. Lancaster 10.1. Introduction 179 10.2. Preparatory Steps 181 10.2.1. Growth of Wolinella succinogenes 181 10.2.2. Isolation of Quinol:Fumarate Reductase 182 10.3. Crystallization of Quinol:Fumarate Reductase 183 10.4. Characterization of the Crystals 185 10.5. Structure Determination 187 11. Crystallization of the Respiratory Complex Formate Dehydrogenase-N from Escherichia coli 193 Mika Jormakka 11.1. Introduction 195 11.2. Expression and Purification of Fdn-N196 11.3. Crystallization and X-ray Data Collection of Fdn-N196 11.4. Structure Determination of Native Fdh-N198 11.5. Determination of the Quinone Binding Site 200 12. Crystallization of the Cytochrome bc1 Complex 203 Li-Shar Huang, David Cobessi, and Edward A. Berry 12.1. About the Protein 205 12.2. Purification Procedures 206 12.3. Early bc1 and b6f Crystallization Studies 207 12.4. Three-Dimensional Crystals of Mitochondrial Cytochrome bc1208 12.5. Large Tetragonal Crystals from the Yu Preparation 209 12.6. Monoclinic, Tetragonal, and Hexagonal Crystals from the Rieske Preparation 210 12.7. Crystallization of the bc1 Complex from Various Vertebrate Organisms in Berkeley 211 12.8. Improved Beef bc1 Crystals from the Jap Group at Berkeley and the Iwata Group in Uppsala 213 12.9. Higher Resolution Crystals of the Fungal bc1 Complex from MPI-Frankfurt 213 12.10. Unpublished Observations from the Berkeley Group 214 12.10.1. Needle Crystals of the Bovine bc1 Complex 214 12.10.2. Hexagonal Bipyramid Crystals (P6522) 215 12.10.3. Conditions for Growth 216 12.10.4. Hexagonal Bipyramid Crystals without Seeding 217 12.10.5. Precrystallization 218 12.10.6. Rabbit Cytochrome bc1 Crystals 218 12.10.7. Orthorhombic Chicken bc1 Crystals 219 12.11. Conclusions 221 13. Crystallization of Cytochrome bo3 Ubiquinol Oxidase from E.coli 227 Jeff Abramson, Bernadette Byrne, and So Iwata 13.1. Purification, Crystallization, and X-Ray Data Collection for Cytochrome bo3229 13.1.1. Crystal Form 1: Wild-Type Cytochrome bo3 229 13.1.2. Crystal Form 2: Cytochrome bo3-Fusion Complex 233 13.2. Structure Determination of Cytochrome bo3235 13.2.1. Structure Determination for Crystal Form 1: Wild-Type Cytochrome bo3 235 13.2.2. Structure Determination and Interpretation for Crystal Form 2: Cytochrome bo3-Protein Z Fusion 236 C. Channel and Receptor 239 14. Crystallization and Structure Determination of MscL, a Gated Prokaryotic Mechanosensitive Channel 241 R. H. Spencer, G. Chang, R. B. Bass, and D. C. Rees 14.1. Introduction 243 14.2. Target Identification 244 14.3. Protein Expression 245 14.4. Protein Purification 246 14.5. Protein Crystallization 248 14.6. Crystallographic Analysis 248 14.7. Conclusions 250 15. Crystallization of Bovine Rhodopsin, a G Protein-Coupled Receptor 253 Tetsuji Okada 15.1. Introduction 255 15.2. Purification Procedure 256 15.2.1. Membrane Preparation 256 15.2.2. Selective Solubilization 257 15.3. Detail of the Crystallization Experiments 258 15.3.1. Protocol 258 15.3.2. The Case History 259 15.4. Characterization of the Crystals 260 15.5. Summary of the Structural Determination 261 15.6. Remarks 261 D. Outer Membrane Proteins 263 16. Crystallization of Phopholipase A in Two Biological Oligomerization States 265 Arjan Snijder, Thomas Barends, and Bauke W. Dijkstra 16.1. Introduction 267 16.2. Purification 268 16.3. Crystallization 269 16.3.1. Monomeric OMPLA 269 16.3.2. Crystal Packing of Monomeric OMPLA 271 16.3.3. Dimeric OMPLA 273 16.3.4. Crystallization under Oil 274 16.3.5. Crystal Packing of Dimeric OMPLA 279 16.4. Conclusion and General Message 276 Part IV. CRYSTALLIZATION INFORMATICS OF MEMBRANE PROTEINS 279 17. Crystallization Informatics of Membrane Proteins 281 So Iwata 17.1. Introduction 283 17.2. Design of a Kit for Membrane Protein Crystallization 284 17.2.1. Selection of Precipitants 286 17.2.2. Selection of Buffers 288 17.2.3. Selection of Salts 288 17.2.4. Design of the Screening Kit 291 17.3. User Instruction of the Kit 291 17.3.1. Protein Concentration 292 17.3.2. Selection of Detergent 292 17.3.3. Sample Buffer 293 17.3.4. Temperature 294 17.3.5. Additives 294 17.3.6. Observation 294 17.4. Summary 294 APPENDICES 299 Questionnaire 302 A1. Porin (OmpF) from Escherichia coli304 A2. Porin from Rhodobacter capsulatus305 A3. Porin from Rhodopseudomonas blastica306 A4. Porin from Paracoccus denitrificans307 A5. Porin Omp32 from Comamonas acidovorans308 A6. Phosphoporin from Escherichia coli 309 A7. Osmoporin from Escherichia coli310 A8. Osmoporin from Klebsiella pneumoniae311 A9. Maltoporin from Escherichia coli312 A10. Maltoporin from Salmonella typhimurium313 A11. Sucroseporin from Salmonella typhimurium314 A12. Exoporin from Escherichia coli315 A13. Siderophore translocator (FhuA) from Escherichia coli316 A14. Siderophore translocator (FhuA) from Escherichia coli317 A15. Siderophore translocator (FepA) from Escherichia coli318 A16. Truncated transmembrane domain of OmpA-protein from Escherichia coli319 A17. OmpX-protein from Escherichia coli320 A18. Phospholipase A from Escherichia coli outer membrane, monomer 321 A19. Phospholipase A from Escherichia coli outer membrane, dimer 322 A20. MscL from Escherichia coli 323 A21. Photosystem I 324 A22. Cytochrome c oxidase from Paracoccus denitrificans325 A23. Cytochrome bc1 complex, P65 form 326 A24. Cytochrome bc1 complex, P6522 form 327 A25. Ubiquinol oxidase from Escherichia coli328 A26. Formate dehydrogenase from Escherichia coli329 A27. Succinate dehydrogenase from Escherichia coli330 A28. Cytochrome c oxidase from Rhodobacter sphaeroides331 A29. Halorhodopsin 332 A30. Bacteriorhodopsin 333 Index 339
I am often asked by people what the trick is to be so successful in obtaining membrane protein crystals. My answer is always “No trick is the trick”. Nobody has ever succeeded in producing a magic solution for membrane protein crystallisation. Therefore, I do not stick to one particular method but try all possible approaches. This flexibility, together with some confidence, may be keys to success. Unfortunately, this determination itself cannot help you to cover the whole multidimensional space of screening conditions of membrane proteins. Therefore, we need a clear strategy to cover the most relevant conditions as efficiently as possible. I believe that understanding the principles of membrane protein crystallisation and learning from successful examples are the key to more rational and efficient screening. Based on this policy, this book was edited. I collected as many successful examples of membrane protein crystallisation as possible and provided chapters to summarize these results. Also included are chapters dealing with the basic principles of membrane proteins such as phase diagrams and classification of membrane protein crystals. I hope this combination is useful for designing your crystallisation experiment. My last word of advice for people starting membrane protein crystallisation is “Think hard and work hard”.
I would thank all the colleagues in my laboratory for their help in editing this book. In particular the scientific contributions and language skills of Dr. Bernadette Byrne have been essential for editing this book. I would also thank all the authors who generously provided their contributions and have been patient during the whole editorial process. Mrs. Terese M. Bergfors at Uppsala University initially recommended me to Dr. Igor Tsigelny at IUL and without them, this book would never be materialized. I must also mentions that all my former supervisors provided me with all the skills necessary to edit this book; they are namely Profs. Takahisa Ohta, Noriyoshi Sakabe, Hartmut Michel, and Janos Hajdu. I would extend my heartfelt gratitude to my wife Momi and my mother Asako for all their support through my entire academic carrier including editing this book. Finally, I dedicate this book to my late father, Isao.
This book is a basic, example-based laboratory manual for scientists and students who crystallize membrane proteins. The book could be useful for wide audience. Using the book, beginners and students find the practical direction how to start; intermediates can improve their techniques; experts can find advisable hints for creating new methods. The book includes four parts. The first part is an introduction in membrane protein chemistry. The second part covers the principles and technique in membrain protein crystallization. The third part displays the examples of successful crystallization of membrane proteins. It conteins three sections: Crystallization of Photosystem I, Respiratory Complexes, and Channel and Receptor. The fourth part is about crystallization informatics of membrane proteins.
Written by the best experts in membrane protein crystallization from different countries.
Edited by So Iwata, Imperial College London, United Kingdom.
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