Experimental Approaches of NMR Spectroscopy (eBook)

The Nuclear Magnetic Resonance Society of Japan was officially constituted on April 1st , 2002. The society promotes the basic and applied studies on nuclear magnetic resonance as well as enlightenment and instructional activity. The society also performs annually a necessary academic meeting and publication of the bulletin, and other service for the achievement. As a part of the activity of the society, it design publication of experimental approaches of NMR spectroscopy including application to life and material sciences.  

Member of the Editorial Board 5
Preface 6
Acknowledgements 8
Contents 9
Methodology 11
1 Protein Studies by High-Pressure NMR 12
Abstract 12
1.1 Introduction 12
1.1.1 The NMR Spectroscopy and Its Limitation 13
1.1.2 Overcoming the Limitation with Pressure 15
1.1.3 Turning NMR “Invisible” Conformers into NMR “Visible” with Pressure 16
1.2 The Thermodynamic Background 19
1.2.1 Effect of Pressure on the Protein Conformational Equilibrium 19
1.2.2 “Volume” Decreases as “Cavity” Hydration Increases 21
1.2.3 The “Volume ??Theorem”?? of Protein and the High-Pressure NMR Experiment 23
1.3 Apparatus for High-Pressure NMR Experiments 24
1.3.1 The Autoclave Method 25
1.3.2 The Pressure-Resisting Cell Method 25
1.4 Application to Protein Studies 28
1.4.1 Fluctuations within the Basic Folded Ensemble 29
1.4.1.1 Fluctuation in Individual Hydrogen Bonds Detected by 1H Pressure Shifts 30
1.4.1.2 Residue-Specific Fluctuations of the Polypeptide Chain Conformation Detected by 15N/1H Pressure Shifts 32
1.4.1.3 Slow Cooperative Fluctuations Detected by Ring-Flip Motions 34
1.4.2 Fluctuations into Alternately Folded Conformer 35
1.4.3 Conformational Fluctuations Involving Fibril Formation and Dissociation 35
1.4.3.1 Cause for Familial Amyloidotic Polyneuropathy 36
1.4.3.2 Conformational Fluctuations in Prion Protein and Drugs to Prevent Fibrillation 37
1.4.3.3 Amyloid Fibril Is a High-Volume State 39
1.4.3.4 Fibril Formation Is Part of the Intrinsic Conformational Fluctuation of Proteins 39
1.4.4 Exploring the Protein Folding Pathway with High-Pressure NMR 40
1.5 Summary: Perspectives of High-Pressure NMR Spectroscopy 41
References 42
2 Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future 46
Abstract 46
2.1 Historical Background of Our Research to Develop Isotope-Aided NMR Methods 47
2.1.1 Stereo-Specific Deuteration of Prochiral Methylene Protons—Conformational Analysis of Amino Acids and Peptides 48
2.1.2 Selective 13C, 15N Double-Labeling Method for the Sequential Assignment of Backbone Amide NMR Signals in Large Proteins 49
2.1.3 Revisiting the Stereo-Specific Isotope-Labeling Approach for Studying Proteins: A Long March to the SAIL Method 51
2.2 The SAIL Method: An Optimized Isotope-Labeling Strategy for the Structural Study of Proteins by NMR Spectroscopy 51
2.2.1 Cell-Free Expression and NMR Spectra of SAIL Proteins 53
2.2.2 Structural Determination of SAIL Proteins 56
2.3 Recent Trends in the Isotope-Aided NMR Methods for Studying Proteins 58
2.3.1 Residue- and Stereo-Specific Labeling Method: The Case for Leu and Val Methyl Labeling of Larger Proteins 59
2.3.2 Large-Amplitude Dynamics of Proteins as Probed by Aromatic Ring-Flipping Motions—The Case for the Interface Between FKBP and Drug Complexes 61
2.3.3 Deuterium-Induced Isotope Shifts for Measuring Hydrogen Exchange Rates of Polar Side-Chain Groups in Proteins: Facile Screening of the Polar Groups Involved in Hydrogen Bond Networks 64
2.4 Future Perspectives of the Isotope-Aided NMR Method 66
Acknowledgements 66
References 67
3 Advances in NMR Data Acquisition and Processing for Protein Structure Determination 71
Abstract 71
3.1 Advances in Processing of Multi-dimensional NMR Spectra, and Their Application to Rapid NMR Measurements 72
3.1.1 Multi-dimensional NMR Takes Time 72
3.1.2 Processing of Multi-dimensional NMR Spectra 73
3.1.3 Rapid Measurement of Multi-dimensional NMR Spectra 75
3.1.4 Non-uniform Sampling 76
3.1.5 NUS Sampling Schemes 77
3.1.6 Semi-constant-Time Evolution Periods 79
3.1.7 Conclusions 82
3.2 Data Analysis for Protein Structure Determination 82
3.2.1 Chemical Shift Data Analysis 82
3.2.2 NOE Data Analysis 85
3.2.3 NMR Protein Structure Determination Based on Bayesian Inference 88
3.2.4 Hybrid Method with Small-Angle Scattering (SAS) 92
3.2.5 Conclusions 94
Acknowledgements 94
References 95
4 Advances in High-Field DNP Methods 99
Abstract 99
4.1 Introduction 100
4.2 Overview of a DNP-NMR System 101
4.3 DNP Mechanisms and Polarizing Agents 101
4.4 SMMW Sources and Transmission Systems 107
4.4.1 Various Light Sources and Gyrotron 107
4.4.2 Principles of Gyrotron 109
4.4.3 Operation of Gyrotron 112
4.4.4 Transmission of SMMW 116
4.4.5 Feedback Regulation of SMMW 117
4.4.6 Double SMMW Irradiation 119
4.5 DNP-NMR Probes and Low-Temperature Facilities 120
4.5.1 MAS at Cryogenic Temperatures 120
4.5.2 SMMW Irradiation of Sample 122
4.5.3 Production of Cryogenic Spinner Gas 125
4.6 DNP Samples and Recent Applications 128
4.6.1 Basic Setup 128
4.6.2 Sample Preparation and DNP Efficiency 130
4.6.3 DNP Enhancement Factor and Temperature 134
4.7 Summary and Outlook 135
Acknowledgements 136
References 136
5 Photoirradiation and Microwave Irradiation NMR Spectroscopy 143
Abstract 143
5.1 Introduction 144
5.1.1 Photoirradiation Solid-State NMR Spectroscopy 144
5.1.2 Microwave Irradiation NMR Spectroscopy 145
5.2 Experimental Details for Photoirradiation Solid-State NMR Spectroscopy 146
5.2.1 Photoirradiation System for Solid-State NMR 146
5.2.2 Photoirradiation NMR Measurements 146
5.2.3 Detection of Photo-Intermediates in the Photoreaction Cycles 147
5.3 Photoreaction Cycle for SRI as Revealed by In Situ Photoirradiation Solid-State NMR 148
5.4 The Photoreaction Cycle of Pharaonis Phoborhodopsin (SRII) as Revealed by Photoirradiation Solid-State NMR Spectroscopy 152
5.5 The Photoreaction Pathway for the Bacteriorhodopsin Y185F Mutant 154
5.6 Experimental Details for Microwave Irradiation NMR Spectroscopy 156
5.6.1 In Situ Microwave Irradiation NMR Spectrometer 156
5.6.2 Temperature Measurements 157
5.6.3 Microwave Temperature Jump Experiments 158
5.7 Microwave Heating Effect of MBBA 159
5.7.1 Microwave Heating Effect of MBBA in the Liquid Crystalline State 159
5.7.2 Microwave Heating Effect of MBBA in the Isotropic State 160
5.7.3 Mechanism for Microwave Heating of Liquid Crystalline MBBA 162
5.7.4 Mechanism for Microwave Heating of Isotropic Phase MBBA 163
5.8 Experimental Details for SC-2D NMR Spectroscopy 164
5.9 SC-2D NMR Spectra of Liquid Crystalline Samples 165
5.9.1 SC-2D NMR Measurements of APAPA 165
5.9.2 Interpretation of the Cross-sectional Spectra from SC-2D NMR Experiments 168
5.9.3 Interpretation of the Dipolar Patterns of the Aromatic and Methyl Protons 170
5.9.4 Interpretation of the Cross-Sectional Patterns 171
5.10 Conclusions 173
Acknowledgements 174
References 174
6 Solid-State NMR Under Ultrafast MAS Rate of 40–120 kHz 179
Abstract 179
6.1 Overview 179
6.2 Setup of Sample-independent Factors 182
6.2.1 Shimming 182
6.2.2 Magic-Angle Adjustment 184
6.2.3 rf Field Strength Calibration 185
6.2.4 Reference 187
6.3 Sample-Dependent Setup 188
6.3.1 Relaxation Delay 188
6.3.2 Hardware Treatment 191
6.4 Setup of 2D Measurements 192
6.4.1 1H DQ/SQ Correlation 192
6.4.2 1H-detected 1H/X CP-HSQC 196
6.5 Conclusions 198
Acknowledgements 199
References 199
7 Elucidating Functional Dynamics by R1? and R2 Relaxation Dispersion NMR Spectroscopy 204
Abstract 204
7.1 Relaxation Dispersion 205
7.2 Accessible Information 206
7.3 R1? Relaxation Dispersion 207
7.3.1 General Aspects 207
7.3.2 Pulse Sequence of the R1? Relaxation Dispersion Experiment 208
7.3.3 Automation of R1? Relaxation Dispersion Measurements and Data Processing 211
7.3.3.1 Experimental Setup 211
7.3.3.2 Screening 212
7.3.3.3 Processing 213
7.4 R2 Relaxation Dispersion 214
7.4.1 General Aspects 214
7.4.2 Quantifying Protein–Ligand Interactions by R2 Relaxation Dispersion 215
7.4.2.1 Theory 216
7.4.2.2 Example 1: Interaction Between the pKID Domain of CREB and the KIX Domain of CBP/p300 217
7.4.2.3 Example 2: Interaction Between the Transactivation Domain of c-Myb and KIX 219
7.5 Fitting of the Relaxation Rates to a Theoretical Model 219
7.5.1 Least-Squares Fitting in GLOVE 219
7.5.2 Monte Carlo Minimization Algorithm in GLOVE 221
7.5.3 Two-State Exchange 221
7.5.4 Workflow for Processing Relaxation Dispersion Data in GLOVE 223
7.5.5 Examples of Relaxation Dispersion Curve Fitting by GLOVE 224
7.6 Outlook 226
References 227
8 Structural Study of Proteins by Paramagnetic Lanthanide Probe Methods 233
Abstract 233
8.1 Introduction 234
8.2 Structural Information Obtained from Paramagnetic Lanthanide Probe Methods 234
8.2.1 Pseudocontact Shift (PCS) 235
8.2.2 Residual Dipolar Coupling (RDC) 235
8.2.3 Paramagnetic Relaxation Enhancement (PRE) 237
8.3 Various Magnetic Properties of Lanthanide Ions 238
8.4 Application of the Paramagnetic Lanthanide Probe in Protein Structural Studies 239
8.4.1 Caged Lanthanide NMR Probe 5 (CLaNP-5) 240
8.4.2 Two-Point Anchored Lanthanide-Binding Peptide Tag (LBT) 241
8.5 Measurement and Analysis of Anisotropic Paramagnetic Effects 245
8.6 Use of Paramagnetic Effects in Protein Structural Studies 246
8.6.1 PCS-Based Docking for Protein–Protein Complexes 246
8.6.2 Evaluation of the Conformational Changes in a Multi-domain Protein 248
8.6.3 Further Applications of Paramagnetic Lanthanide Probe Methods 249
8.7 Concluding Remarks 251
Acknowledgements 252
References 252
9 Structure Determination of Membrane Peptides and Proteins by Solid-State NMR 259
Abstract 259
9.1 Introduction 260
9.2 Experimental Approaches Used in Solid-State NMR Spectroscopy 260
9.2.1 Experimental Details for Obtaining the Structures of Membrane-Associated Peptides and Proteins Using Anisotropic Interactions 261
9.2.1.1 Orientation Dependence of Chemical-Shift Interaction 261
9.2.1.2 Orientation Dependence of Nuclear Dipolar Interactions 266
9.2.1.3 Interatomic Distance Measurements by REDOR 267
Simple Description of the REDOR Experiment 267
Rotational Echo Amplitude by the Density Operator Approach 269
Echo Amplitude in Three-Spin System (S1-I1-S2) 271
Practical Aspects of a REDOR Experiment 272
9.2.2 Magic-Angle Spinning NMR 274
9.2.2.1 CP-MAS NMR 274
9.2.2.2 Correlation NMR Spectroscopy 275
9.2.2.3 PDSD and DARR 2D NMR Spectroscopy 275
9.3 Structure Determination of Membrane-Bound Peptides 277
9.3.1 Melittin 277
9.3.2 Alamethicin 283
9.3.3 Bovine Lactoferrampin 284
9.4 Structure Determination of Membrane Proteins 287
9.5 Conclusion 291
Acknowledgements 293
References 293
Application to Life Science and Materials Science 300
10 NMR Studies on Silk Materials 301
Abstract 301
10.1 Introduction 301
10.2 Results and Discussion 302
10.2.1 Dynamics of Silk Fibroin Stored in Living Silkworm 302
10.2.2 Solution Structure of Silk Fibroin at Atomic Level 304
10.2.3 Dynamics of Water Molecules Interacted with Silk Fibroin 305
10.2.4 Dynamics of Hydrated Silk Cocoon, Sericin, and Fibroin 307
10.2.5 Fraction of Several Conformations of Silk Fibroin 308
10.2.6 Domain of Silk Fibroin 309
10.2.7 Inter-molecular Arrangement of Alanine Oligopeptide 310
10.2.8 Complex between Silk Fibroin and Glycerin 310
10.2.9 Use of Chemical Shift Calculation for Verification of Silk Fibroin Structural Model 312
10.3 Conclusion 313
References 314
11 NMR Studies on Polymer Materials 317
Abstract 317
11.1 Introduction 318
11.2 Polymer Blends and Alloys 318
11.2.1 Miscibility and Mobile Heterogeneity 318
11.2.1.1 PS/PVME Blends 320
11.2.1.2 PMAA/PVAc Blends 323
11.2.1.3 PK/PA Alloys 325
11.2.2 Phase Separation 328
11.2.2.1 PS/PVME Blends 328
11.2.2.2 P3HT/PCBM Blends 330
11.3 Polymer Nanocomposites 332
11.3.1 Paramagnetic Effect on Relaxation 332
11.3.1.1 PVA/Montmorillonite Clay Nanocomposites 332
11.3.1.2 Nylon-6/mmt Nanocomposites 333
11.3.2 Interaction Between Polymers and Fillers 334
11.3.2.1 Nylon-6/Montmorillonite Clay Nanocomposites 334
11.3.2.2 PS-PEO Block Copolymer/Hectorite Clay Nanocomposites 335
11.3.3 Morphology 336
11.3.3.1 PVIBE/?-PL/Saponite Clay Blends 336
11.3.3.2 Nylon-6/Montmorillonite Clay Nanocomposites 337
11.4 Rubbers and Elastomers 339
11.5 Conclusion 340
References 341
12 Solid-State 2H NMR Studies of Molecular Motion in Functional Materials 344
Abstract 344
12.1 Introduction 345
12.2 Measurements of Solid-State 2H NMR Spectrum 345
12.3 Simulation Methods of Solid-State 2H NMR Spectrum 347
12.4 Analysis of MOF/PCP 354
12.5 Analysis of Solid Proton-Conducting Material 356
12.6 Analysis of Spin-Crossover Material 361
12.7 Summary 365
References 366
13 NMR Spectral Observations of the Gases in Polymer Materials 368
Abstract 368
13.1 Introduction 368
13.2 Basics of the NMR Analysis of Gas–Polymer Systems 369
13.2.1 Apparatus for the Preparation of NMR Sample Tubes Containing High-Pressure Gas Samples 369
13.2.2 Gas Sorption Properties of Polymers 370
13.3 Characterization of the High-Order Structure of a Glassy Polymer Based on 129Xe NMR Chemical Shifts 372
13.4 Characterization of the High-Order Structure of Rubbery Polymers from 129Xe NMR Chemical Shifts 374
13.5 Analysis of Gas Diffusion Characteristics in Polymers Based on NMR Peak Width 376
13.6 Characterization of Oriented Structures by Pulsed Field Gradient NMR 379
13.7 Summary 382
References 382
14 NMR Studies on Natural Product—Stereochemical Determination and Conformational Analysis in Solution and in Membrane 385
Abstract 385
14.1 Stereochemical Determination of Natural Products 386
14.1.1 JBCA Method 387
14.1.2 UDB Method 392
14.1.3 Calculations 394
14.1.4 RDCs 398
14.2 NMR Methods for Examining the Conformation and Intermolecular Interactions of Natural Products in Membranes 401
14.2.1 Amphotericin B 402
14.2.2 Erythromycin A 406
14.2.3 Theonellamide A 407
14.3 Summary and Outlook 412
Acknowledgements 412
References 412
15 Technical Basis for Nuclear Magnetic Resonance Approach for Glycoproteins 417
Abstract 417
15.1 Introduction 418
15.2 Enigmatic Aspects of Carbohydrate Structures 418
15.3 Expression of Isotope-Labeled Glycoproteins 420
15.4 Glycosylation Profiling 425
15.5 Remodeling of Glycoprotein Glycoforms 426
15.6 Spectral Observations and Assignments 430
15.7 NMR Analyses of Dynamic Conformations and Interactions of Oligosaccharides 432
15.8 Perspectives 434
Acknowledgements 435
References 435
16 NMR Studies on RNA 441
Abstract 441
16.1 Design of RNA Sequences 441
16.2 Sample Preparation 442
16.2.1 In Vitro Transcription 444
16.2.2 Chemical Synthesis 444
16.2.3 Enzymatic Ligation 445
16.2.4 Artificial Base Pair System 445
16.2.5 Stable Isotopic Labelling 446
16.2.6 Purification 446
16.3 Measurements 447
16.3.1 Exchangeable Protons 447
16.3.2 Non-exchangeable Protons 449
16.3.3 Residual Dipolar Coupling 449
16.4 Signal Assignments 451
16.4.1 Exchangeable Protons 451
16.4.2 Non-exchangeable Protons 451
16.5 Structural Calculation 455
16.6 Interaction Analysis and Structure Screening 457
16.7 Perspective 459
References 459
17 NMR Analysis of Molecular Complexity 462
Abstract 462
17.1 Introduction 463
17.2 Metabolomics and Metabolic Profiling for Small Molecular Complexity 463
17.2.1 Basic Knowledge for Small Molecular Profiling 463
17.2.2 Experimental Aspects of Sample Preparation 464
17.2.3 NMR Measurements and Data Processing 466
17.2.3.1 Useful Pulse Sequences 466
17.2.3.2 Databases and Tools for NMR Analysis 467
17.2.3.3 Signal Assignments and Structure Elucidation 467
17.2.3.4 Spectral Pretreatment for Data Science 470
17.2.3.5 Practical Aspects of Data Science 471
17.2.4 Applications to Plant, Animal, and Microbial Systems 471
17.2.4.1 Application to Plant Systems 471
17.2.4.2 Application to Animal Systems 472
17.2.4.3 Application to Microbial Systems 473
17.3 Biomass Profiling for Macromolecular Complexity 474
17.3.1 Basic Knowledge for Macromolecular Profiling 474
17.3.2 Experimental Aspects of Sample Preparation 474
17.3.3 NMR Measurements and Data Processing 476
17.3.3.1 Useful Pulse Sequences 476
Peak Separation for Broad Macromolecular Spectra 478
17.3.4 Applications to Material, Biological, and Geochemical Systems 479
17.3.4.1 Application to Cellulosic Material Systems 479
17.3.4.2 Application to Biological Systems 480
17.3.4.3 Application to Geochemical Samples 482
17.4 Future Perspectives 483
References 483
18 NMR of Paramagnetic Compounds 491
Abstract 491
18.1 Introduction 491
18.2 Paramagnetic Effects 494
18.2.1 Paramagnetic Shifts 494
18.2.2 Paramagnetic Relaxation 495
18.3 1H NMR Spectra of Myoglobin with 0, 1, 4, or 5 Unpaired Electrons 498
18.3.1 Overview 498
18.3.2 Diamagnetic Carbonmonoxy Form [Mb(CO)] 499
18.3.3 Paramagnetic Deoxy Form (Deoxy Mb) with S = 2 499
18.3.4 Paramagnetic Met-Cyano Form [MetMb(CN?)] with S = ½ 502
18.3.5 Paramagnetic Met-Azido Form [MetMb(N3?)] with Mainly S = ½ 504
18.3.6 Paramagnetic Met-Aquo Form [MetMb(H2O)] with S = 5/2 505
18.4 NMR Measurements 506
18.4.1 NOEs in Paramagnetic Compounds 506
18.4.2 Hydrogen Exchange Rates 509
18.5 Concluding Remarks 512
Acknowledgements 513
References 513
19 NMR of Quadrupole Nuclei in Organic Compounds 519
Abstract 519
19.1 Introduction 519
19.2 Theoretical Background of Quadrupole Interactions 520
19.3 Simulating Solid-State NMR of Quadrupole Nuclei Based on the Perturbation Method 522
19.3.1 Stationary NMR Spectra Under the Influence of Second-Order Quadrupole Interactions 524
19.3.2 Fast MAS NMR Conditions Under the Influence of Second-Order Quadrupole and CS Interactions 527
19.3.3 Stationary NMR Spectra Under the Influence of Second-Order Quadrupole and CS Interactions 530
19.4 Solid-State NMR of Quadrupole Nuclei Based on Direct Calculation Methods 534
19.5 Conclusions 541
References 542
20 Quadrupole Nuclei in Inorganic Materials 544
Abstract 544
20.1 Introduction 545
20.2 Anisotropy of Quadrupolar Interaction 546
20.3 Orientation Dependence of Quadrupolar Interaction 552
20.4 Signal Separation Through Quadrupolar Coupling 557
20.4.1 MQMAS 558
20.4.2 STMAS 561
20.5 Signal Separation Through Correlations to Other Nuclei 561
20.6 Sensitivity Enhancement Through Population Transfer 565
20.7 Confronting the Real World, Real Materials 567
Acknowl?edge??ments 572
References 572
21 Protein–Ligand Interactions Studied by NMR 577
Abstract 577
21.1 Overview 578
21.2 Ligand-Based Approach 578
21.2.1 Ligand Screening 581
21.2.1.1 Saturation Transfer Difference (STD) 581
21.2.1.2 WaterLOGSY 581
21.2.1.3 STD Versus WaterLOGSY 582
21.2.2 Pharmacophore Determination 582
21.2.2.1 DIRECTION 583
21.2.2.2 INPHARMA 583
21.2.3 Structural Information-Driven Ligand Design 584
21.2.3.1 Inter-ligand Nuclear Overhauser Effect (ILOE) 585
21.2.3.2 Transferred Nuclear Overhauser Effect (trNOE) 585
21.2.3.3 Technical Comments on INPHARMA, ILOE and trNOE Experiments 586
21.3 Protein-Based Approach for Protein–Ligand Interactions 586
21.3.1 Ligand Screening and NMR-Assisted Fragment-Based Drug Discovery 588
21.3.1.1 HSQC-Based Ligand Screening 588
21.3.1.2 SAR-by-NMR 589
21.3.2 Determination of the Molecular Interface and Exploring the Mode of Action 589
21.3.2.1 Chemical Shift Mapping 589
21.3.2.2 KD Determination by the NMR Titration 590
21.4 Experimental Aspects of the NMR Study of Protein–Ligand Interactions 591
21.4.1 Stable Isotope Labeling 591
21.4.2 Tips for the NMR Titration Experiment 592
21.4.2.1 Sample Handling 592
21.4.2.2 Data Acquisition and Analysis 594
21.5 Other Complementary Methods for Protein–Ligand Interactions 594
21.6 Perspective 595
References 595
22 Protein Structure and Dynamics Determination by Residual Anisotropic Spin Interactions 599
Abstract 599
22.1 Why Do We Need Residual Anisotropic Nuclear Spin Interactions in Solution NMR? 600
22.2 Theoretical Backgrounds of the Residual Anisotropies 602
22.2.1 Residual Dipolar Coupling, RDC 603
22.2.2 Theoretical Description on RDC 605
22.2.3 Residual Chemical Shift Anisotropy, RCSA 608
22.3 Practical Procedures to Use the Residual Anisotropies 611
22.3.1 Magnetically Aligning Liquid Crystalline Media 611
22.3.2 Naturally Occurring Materials that Spontaneously Align in a Magnetic Field 613
22.3.3 Compressed Acryl Amide Gel 613
22.3.4 Protein Structures in Weakly Aligned Media 615
22.4 RDC-Based Domain Orientation Analysis 616
22.4.1 Collecting the RDC Data 617
22.4.2 Domain Orientation Analysis Based on the RDC Data 617
22.4.3 Significance of the Domain Orientation Analysis by RDCs 619
22.4.4 Molecular Size Limitation in the RDC-Based Approach 619
22.4.5 Existing Remedy for Overcoming the Size Limitation in the RDC-Based Approach 621
22.5 Alignment Tensor Determination Using Only TROSY 622
22.5.1 Alignment-Induced TROSY Shift Changes 622
22.5.2 CSA Tensor Parameters Used in DIORITE 624
22.5.3 DIORITE Analysis Using Different Magnetic Field Strengths 625
22.5.4 Practical Aspects of the DIORITE Data Collection 627
22.5.5 DIORITE Analyses on MBP in Different Ligand Bound States 628
22.6 Conclusion 630
References 631