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-N     196
     11.3. Crystallization and X-ray Data Collection of Fdn-N     196
     11.4. Structure Determination of Native Fdh-N     198
     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 bc1     208
     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 bo3     229
              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 bo3     235
              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 coli     304
A2. Porin from Rhodobacter capsulatus     305
A3. Porin from Rhodopseudomonas blastica     306
A4. Porin from Paracoccus denitrificans     307
A5. Porin Omp32 from Comamonas acidovorans     308
A6. Phosphoporin from Escherichia coli     309
A7. Osmoporin from Escherichia coli     310
A8. Osmoporin from Klebsiella pneumoniae     311
A9. Maltoporin from Escherichia coli     312
A10. Maltoporin from Salmonella typhimurium     313
A11. Sucroseporin from Salmonella typhimurium     314
A12. Exoporin from Escherichia coli     315
A13. Siderophore translocator (FhuA) from Escherichia coli     316
A14. Siderophore translocator (FhuA) from Escherichia coli     317
A15. Siderophore translocator (FepA) from Escherichia coli     318
A16. Truncated transmembrane domain of OmpA-protein from Escherichia coli     319
A17. OmpX-protein from Escherichia coli     320
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 denitrificans     325
A23. Cytochrome bc1 complex, P65 form     326
A24. Cytochrome bc1 complex, P6522 form     327
A25. Ubiquinol oxidase from Escherichia coli     328
A26. Formate dehydrogenase from Escherichia coli     329
A27. Succinate dehydrogenase from Escherichia coli     330
A28. Cytochrome c oxidase from Rhodobacter sphaeroides     331
A29. Halorhodopsin     332
A30. Bacteriorhodopsin     333
Index     339
 

Preface

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”.




Acknowledgements

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.


So Iwata
London