PREFACE ..................................................... xxiii
FOREWORD ...................................................... xxv
ACKNOWLEDGEMENTS ............................................ xxvii
ABBREVIATIONS ................................................ xxix
CHAPTER 1: BASIC CONCEPTS IN NMR SPECTROSCOPY ................... 1
1 HISTORICAL PERSPECTIVES ...................................... 1
1.1 Quantum Mechanical Model for Spin-1/2 Nuclei ............ 2
1.2 Classical Model ......................................... 4
1.2.1 Rotating Frame of Reference ...................... 5
1.2.2 Strength of RF Pulses ............................ 5
1.3 Basic Design of a NMR Spectrometer ...................... 6
1.4 A Simple Pulse Sequence for NMR Excitation and
Detection ............................................... 8
1.5 Fourier Transform ....................................... 9
1.6 Line Widths ............................................ 10
1.7 Properties of Spin Operators and Pauli Matrices ........ 10
2 NMR SPINS USED IN LIFE SCIENCES ............................. 11
3 INTERACTION OF NUCLEAR SPINS AND NMR PARAMETERS ............. 12
3.1 Chemical Shift (δ) ..................................... 12
3.2 Peak Intensities ....................................... 15
3.3 Nuclear Spin-Spin (Scalar) Coupling (J) ................ 16
3.4 Hamiltonian for α 2-spin System ........................ 17
3.5 Dipolar Coupling (Dij) ................................. 20
3.6 Quadrupolar Interactions ............................... 21
3.7 Electron-Nuclear Interaction ........................... 22
4 NMR RELAXATION .............................................. 23
4.1 Relaxation Rates ....................................... 23
4.2 Molecular Mechanisms Leading to Relaxation ............. 24
4.3 Theoretical Treatment of Relaxation Rates .............. 25
4.4 Correlation Times ...................................... 26
4.5 Dipolar Relaxation due to Several Interacting Spins .... 29
4.6 Field Dependence of Relaxation Rates ................... 29
5 CHEMICAL EXCHANGE: DYNAMIC EFFECTS IN NMR SPECTROSCOPY ...... 30
5.1 Conformational Equilibrium ............................. 30
5.2 Solvent Exchange ....................................... 32
6 NUCLEAR MAGNETIC DOUBLE RESONANCE ........................... 33
6.1 Spin Decoupling ........................................ 33
6.2 Heteronuclear and Broad-band Decoupling ................ 34
6.3 Nuclear Overhauser Effect (NOE) ........................ 34
7 LINE SHAPES IN NMR .......................................... 37
7.1 Absorptive and Dispersive Signals ...................... 37
7.2 Characteristics of FT-NMR Signals ...................... 38
7.3 Quadrature Phase Detection ............................. 39
8 REFERENCES .................................................. 40
8.1 History and Early Developments ......................... 40
8.2 Theoretical and Physical Aspects of NMR ................ 40
8.3 Chemistry Oriented Books ............................... 41
8.4 Specialized Topics ..................................... 41
CHAPTER 2: INTRODUCTION TO BIOLOGICAL NMR ...................... 43
1 LEVELS OF BIOLOGICAL STRUCTURES ............................. 44
2 NMR AND BIOLOGICAL STRUCTURES ............................... 46
3 DIFFICULTIES IN STUDYING BIOLOGICAL SYSTEMS BY NMR .......... 47
3.1 Sensitivity ............................................ 47
3.2 Resolution ............................................. 48
3.3 Assignments ............................................ 48
3.4 Water Signal ........................................... 48
3.5 Line Widths ............................................ 49
3.6 Quantification ......................................... 50
4 BIOLOGICAL MACROMOLECULES ................................... 50
4.1 Building Blocks of Biological Molecules ................ 50
4.2 Biopolymers ............................................ 51
4.3 3D Structures of Biological Molecules .................. 52
4.4 Comparison of 3D Structures Obtained from NMR and
X-ray .................................................. 53
5 NMR IN CELLS AND TISSUES .................................... 54
5.1 Cellular Metabolism is the Bridge between Proteomics
and Function ........................................... 55
6 NMR IN STUDIES OF ORGANS .................................... 55
6.1 Historical Development of MRI .......................... 56
6.2 Basis of MRI ........................................... 56
6.3 Comparison of Images from NMR and Other Techniques ..... 57
6.4 Magnetic Resonance Spectroscopy (MRS) .................. 59
7 BASIC MULTI-PULSE NMR EXPERIMENTS IN BIOLOGICAL SYSTEMS ..... 59
7.1 Signal Averaging and Partial Saturation ................ 59
7.2 Presaturation .......................................... 61
7.3 Jump and Return (JR) Sequence .......................... 61
7.4 Spin-echo (SE) ......................................... 62
7.4.1 Effect of Inhomogeneity of B0 ................... 62
7.4.2 Behaviour of J-Coupled Systems .................. 63
7.4.3 Application of SE ............................... 66
7.5 Carr-Purcell-Meiboom-Gill (CPMG) Sequence .............. 66
7.6 Inversion Recovery Experiment (IR) ..................... 66
8 COMPARISION OF NMR OTHER PHYSICAL TECHNIQUES ................ 67
9 REFERENCES .................................................. 68
9.1 Theoretical and Physical Aspects of NMR ................ 68
9.2 Biological Molecules ................................... 68
9.3 Biomedical Systems ..................................... 68
CHAPTER 3: MULTI-DIMENSIONAL NMR ............................... 69
1 INTRODUCTION ................................................ 69
1.1 Multi-Dimensional (MD) NMR ............................. 69
1.2 General Scheme for 2D NMR .............................. 69
1.3 Correlated Spectroscopy (COSY) ......................... 72
1.4 Advantages of 2D NMR ................................... 73
2 PRODUCT OPERATOR FORMALISM (POF) ............................ 74
2.1 Product Operators (PO) ................................. 75
2.2 Coherences ............................................. 75
2.3 Observable Coherences .................................. 77
2.4 Effect of RF Pulses on Product Operators ............... 77
2.5 Evolution of Product Operators under free
Hamiltonian ............................................ 78
2.6 Effect of Composite я pulses on Product Operators ...... 78
2.7 Evolution of MQ Coherences ............................. 79
2.8 Evolution of Zero Quantum Coherences ................... 79
2.9 Evolution of Two Quantum Coherences .................... 79
3 HOMONUCLEAR CORRELATION SPECTROSCOPY ........................ 80
3.1 Correlation Spectroscopy (COSY) ........................ 80
3.2 Phase Cycling .......................................... 81
3.2.1 CYCLOPS ......................................... 82
3.2.2 Axial Peak Suppression .......................... 82
3.3 Relayed COSY ........................................... 83
3.4 TOtal Correlation Spectroscopy (TOCSY) ................. 84
3.5 MQ-Filtered COSY ....................................... 85
3.5.1 Multiple-Quantum Filtering ...................... 85
3.5.2 Double-Quantum Filtered COSY (2QF-COSY) ......... 86
3.5.3 Three-Quantum Filtered COSY (3QF-COSY) .......... 87
3.5.4 Exclusive COSY .................................. 89
3.6 EXSY and NOESY ......................................... 90
3.7 ROtational Nuclear Overhauser Effect SpectroscopY
(ROESY) ................................................ 91
4 MULTIPLE-QUANTUM (MQ) SPECTROSCOPY .......................... 92
4.1 2D Double-Quantum (DQ) Experiment ..................... 92
5 HETERONUCLEAR CORRELATION SPECTROSCOPY ...................... 94
5.1 Insensitive Nuclei Enhanced by Polarization Transfer
(INEPT) ................................................ 94
5.2 Reverse INEPT .......................................... 96
5.3 Refocused INEPT ........................................ 96
5.4 Distortionless Enhanced by Polarization Transfer
(DEPT) ................................................. 97
5.5 Broadband Decoupling ................................... 97
5.6 Heteronuclear Single-Quantum Correlation Spectroscopy
(HSQC) ................................................. 99
5.7 Heteronuclear Multiple-Quantum Correlation
Spectroscopy (HMQC) ................................... 101
5.8 Sensitivity Enhanced HSQC ............................. 101
6 PULSED FIELD GRADIENTS ..................................... 102
6.1 Coherence Pathway Selection using PFG ................. 103
6.2 Water Suppression by Gradient Tailored Excitation
(WATERGATE) ........................................... 104
7 THREE DIMENSIONAL (3D) EXPERIMENTS ......................... 105
7.1 3D Double-Resonance Experiments ....................... 106
7.2 3D NOESY-[13C/15N-1H] HSQC ............................ 106
7.3 3D TOCSY-[13C/15N-1H] HSQC ............................ 108
7.4 3D Triple Resonance Experiments ....................... 108
8 TECHNIQUES FOR SOLUTION NMR OF VERY LARGE MOLECULES ........ 108
9 REFERENCES ................................................. 110
9.1 Theoretical and Physical Aspects ...................... 110
9.2 Review Articles ....................................... 110
9.3 Three Dimensional NMR ................................. 110
10 APPENDIX 3.1 ............................................... 111
CHAPTER 4: BIOMOLECULAR STRUCTURES USING NMR: GENERAL
PRINCIPLES ......................................... 113
1 INTRODUCTION ............................................... 113
1.1 Conformation of Biological Molecules .................. 114
1.2 Conformational Theory ................................. 115
1.3 Conformational Domains of Proteins and Nucleic
Acids ................................................. 118
2 ELEMENTS OF MACROMOLECULAR STRUCTURES ...................... 119
2.1 Primary Structure ..................................... 119
2.2 Secondary Structures .................................. 120
2.3 Tertiary Structures ................................... 120
2.4 Quaternary Structures ................................. 120
2.5 Multi-molecular Assemblies ............................ 121
2.6 Random Coil Structures ................................ 121
3 SAMPLE PREPARATION FOR NMR: LABELLING TECHNIQUES ........... 121
3.1 Preparation of Samples for NMR ........................ 122
3.1.1 Cloning and Expression of Proteins ............. 123
3.1.2 Taming Proteins ................................ 124
3.1.3 Protein Recovery ............................... 125
3.1.4 Protein Purification ........................... 125
3.1.5 Ion-exchange Chromatography .................... 126
3.1.6 Affinity Chromatography ........................ 126
3.1.7 Gel Filtration ................................. 126
3.1.8 Approaches in Structural Genomics .............. 126
3.2 Isotope Labelling ..................................... 127
3.3 Concentration ......................................... 127
3.4 Quality Assessment and Storage ........................ 128
3.5 Synthesis of Nucleic Acids ............................ 128
3.6 RNA Samples ........................................... 128
3.7 Purification of Nucleic Acids ......................... 129
3.8 13C and 15N Labelling of Nucleic Acids ................. 129
3.9 In-vitro RNA Transcription and Purification ........... 130
4 NMR APPROACH TO STRUCTURAL STUDIES ......................... 130
4.1 NMR Strategies ........................................ 131
4.2 General Approach for the Resonance Assignments in
Biomolecules .......................................... 131
5 NMR PARAMETERS FOR STRUCTURAL STUDIES ...................... 133
5.1 Chemical Shifts: Magnetic Anisotropy of Chemical
Groups ................................................ 133
5.1.1 Ring Current Effects ........................... 134
5.1.2 Changes in Chemical Shifts and Secondary
Structures ..................................... 135
5.2 Chemical Shifts and 2H Exchange Rates of Hydrogen
Bonded Protons ........................................ 135
5.3 Nuclear Overhauser Effect (NOE) ....................... 136
5.3.1 Limitations of NOE ............................. 136
5.3.2 Introduction of NOE Constraints ................ 138
5.4 Scalar Coupling Constants (J) ......................... 138
5.4.1 Torsion Angle Dependence of 3J ................. 139
5.4.2 Coupling Constants across Hydrogen Bonds ....... 140
5.5 Residual Dipolar Couplings (RDC) ...................... 141
5.5.1 Partial Alignment .............................. 142
5.5.2 Methods of Achieving Partial Alignment of
Biological Molecules ........................... 144
5.5.3 Use of RDC for 3D Structure Determination ...... 144
5.5.4 Advantages of RDC .............................. 145
5.6 Use of Cross-Correlated Relaxation .................... 145
5.7 Covalent Bond Distances ............................... 147
5.8 Stereochemical Assignments ............................ 148
5.9 Removing Undesirable Restraints ....................... 149
6 PARAMAGNETIC MOLECULES AND REAGENTS ........................ 149
6.1 Contact Shifts ........................................ 150
6.2 Pseudo-Contact Shifts ................................. 150
6.3 Relaxation Rates ...................................... 151
6.4 Metallo-Proteins ...................................... 152
6.5 Lanthanides Shift Reagents ............................ 152
6.6 Use of Spin Labels .................................... 153
7 FROM NMR PARAMETERS TO STRUCTURES .......................... 153
7.1 Starting Structures ................................... 153
7.2 Metric Matrix Distance Geometry ....................... 154
7.3 Variable Target Functions: Torsion Angle Approaches ... 155
7.4 Molecular Mechanics and Molecular Dynamics
Algorithms ............................................ 155
7.5 Torsion Angle Dynamics (TAD) .......................... 157
7.6 Restraint Energy Minimization ......................... 158
7.7 Validation of Final Structures ........................ 159
7.8 Presenting the Final Structures ....................... 160
8 DATA BANKS FOR STRUCTURES AND NMR OF BIOMOLECULES .......... 160
8.1 Protein and Nucleic Acid Data Banks (PDB) ............. 160
8.2 Biological Magnetic Resonance Data Bank
(BioMagResBank; BMRB) ................................. 161
8.3 Applications of Data Banks ............................ 161
8.4 Where to look for Literature on NMR Structures ........ 162
9 REFERENCES ................................................. 162
9.1 Further Reading ....................................... 162
9.2 Books ................................................. 162
CHAPTER 5: PROTEIN NMR: GENERAL PRINCIPLES AND RESONANCE
ASSIGNMENTS ........................................ 163
1 INTRODUCTION ............................................... 163
1.1 Functions of Proteins ................................. 163
1.2 Conformation and Dynamics of Proteins ................. 164
1.3 History of Protein Structure Determination ............ 166
2 ELEMENTS OF PROTEIN STRUCTURES ............................. 167
2.1 Nomenclature .......................................... 167
2.2 Backbone and Side-chain Torsion Angles ................ 167
2.3 Primary Structure ..................................... 169
2.4 Conformational Freedom of Peptides .................... 170
2.5 Disulfide Bridges and Proline Rings ................... 172
2.6 Secondary Structures .................................. 172
2.6.1 α-helix ........................................ 173
2.6.2 β-strands and β-sheet ......................... 173
2.6.3 β-turns ........................................ 175
2.6.4 Collagen ....................................... 175
2.6.5 Poly (L-Proline) ............................... 175
2.7 Fibrous Proteins ...................................... 176
2.8 Tertiary Structure: Globular Proteins ................. 176
2.9 Higher Levels of Structural Organization .............. 176
3 INTRODUCTION TO NMR OF PROTEINS ............................ 177
3.1 History of NMR of Proteins ............................ 177
3.2 Current Status ........................................ 178
3.3 Sample Preparation .................................... 179
3.4 NMR Approach to Protein Structure ..................... 179
3.5 Classification of Chemical Shifts ..................... 180
3.6 13Cα and 13Cβ Chemical Shift Statistics ................ 181
4 RESONANCE ASSIGNMENT STRATEGIES ............................ 181
4.1 Identification of Networks of J-coupled Spin
Systems ............................................... 181
4.1.1 Glycine ........................................ 182
4.1.2 Amino acids Containing Methyl Group(s): Ala,
Thr, Val, Ile and Leu .......................... 182
4.1.3 Asn, Asp, Cys and Ser .......................... 183
4.1.4 His, Phe, Trp and Tyr .......................... 183
4.1.5 Long Side-chain Residues ....................... 184
4.2 Linking Side Chains to the Respective Amide Protons ... 184
4.3 Sequence Specific Resonance Assignments ............... 184
4.4 Identification of Disulfide Bridges ................... 185
4.5 Stereo-specific Resonance Assignments ................. 186
4.6 Higher Dimensional NMR ................................ 186
5 3D TRIPLE-RESONANCE EXPERIMENTS FOR PROTEINS ............... 186
5.1 HNCA .................................................. 187
5.2 HN(CO)CA .............................................. 189
5.3 HNCO .................................................. 190
5.4 HN(CA)CO .............................................. 190
5.5 CBCANH ................................................ 191
5.6 CBCA(CO)NH ............................................ 191
5.7 HN(CA)HA and HN(COCA)HA ............................... 191
5.8 3D Experiments used for Side-Chain Resonance
Assignments in Proteins ............................... 192
6 REDUCED DIMENSIONALITY: G-MATRIX FTNMR FOR PROTEINS ........ 192
6.1 RDNMR ................................................. 193
6.2 GFTNMR ................................................ 195
7 AUTOMATED NMR ASSIGNMENTS IN PROTEINS ...................... 197
7.1 Tracked Automated Assignment in Proteins (TATAPRO) .... 198
7.2 Selective Labeling/Unlabeling: Residue Specific
Resonance Assignments ................................. 199
7.3 Side-chain Assignments ................................ 200
7.4 Automated Chemical Shift Prediction Based on
'Sequence Homology .................................... 200
8 TECHNIQUES FOR STUDYING LARGE PROTEINS ..................... 201
8.1 Transverse Relaxation Optimized SpectroscopY
(TROSY) ............................................... 201
8.2 2H labelling .......................................... 202
9 PROTONLESS MULTI-DIMENSIONAL NMR ........................... 203
9.1 2D Protonless NMR ..................................... 204
9.2 The Problem of Large Homonuclear Couplings in
Acquisition Dimension ................................. 204
9.3 3D Protonless NMR ..................................... 207
10 REFERENCES ................................................. 208
10.1 Historical Foundation of Protein Structure and
Function .............................................. 208
10.2 General Books on Proteins ............................. 208
10.3 Books on Protein NMR .................................. 209
10.4 Study of Large Proteins ............................... 209
CHAPTER 6: STRUCTURE, DYNAMICS AND FUNCTION OF PROTEINS ....... 211
1 INTRODUCTION ............................................... 211
2 CHARACTERIZATION OF SECONDARY STRUCTURE ELEMENTS ........... 211
2.1 Chemical Shift Index (CSI) ............................ 211
2.2 Secondary Structure using Chemical Shift and
Sequence Homology ..................................... 213
2.3 Nuclear Overhauser Effect (NOE) ....................... 213
2.4 Three bond Scalar-couplings (3J) ...................... 215
2.5 1HN-2HN Exchange ....................................... 215
3 OBTAINING THE FINAL STRUCTURES ............................. 217
3.1 3D Structure Calculation .............................. 217
3.2 Automated NOESY Assignments ........................... 218
3.3 Structural Statistics and Quality ..................... 218
4 PROTEIN DYNAMICS ........................................... 219
4.1 NMR Parameters for Studying Dynamics .................. 220
4.2 Basic Theory .......................................... 220
4.3 Protein Dynamics using NMR ............................ 221
4.4 Experimental Results .................................. 222
5 PROTEIN FOLDING AND UNSTRUCTURED PROTEINS .................. 223
5.1 Protein Folding Pathways .............................. 223
5.2 NMR Methodologies for Studying Unfolded Protein
Structures ............................................ 224
5.3 Molten Globules ....................................... 225
5.4 Unstructured Functional Proteins ...................... 225
5.5 Protein Structures under High Pressure ................ 226
6 NMR STUDIES OF PROTEIN STRUCTURE, DYNAMICS AND FOLDING:
SOME SPECIFIC EXAMPLES ..................................... 226
6.1 Basic Pancreatic Trypsin Inhibitor (BPTI) ............. 227
6.2 Human Ubiquitin ....................................... 228
6.3 Ribonuclease .......................................... 228
6.4 Lysozyme .............................................. 228
6.5 EF-hand Calcium Binding Proteins (CaBP) ............... 230
6.6 Maleate Synthase G (MSG) .............................. 231
6.7 GroEL-GroES Complex ................................... 231
6.8 Membrane Proteins Studied in Solutions ................ 232
7 PARAMAGNETIC PROTEINS ...................................... 232
7.1 Metal Ions of Interest ................................ 233
7.2 Heme Iron Proteins .................................... 234
7.2.1 High-Spin Iron (III) ........................... 236
7.2.2 Low Spin Iron (III) ............................ 237
7.2.3 High Spin Iron (II) ............................ 237
7.2.4 Diamagnetic Iron Heme Proteins ................. 238
7.3 Iron-Sulfur Proteins .................................. 238
7.4 Other Iron Proteins ................................... 239
7.5 Other Metal Ions ...................................... 239
8 FUNCTIONAL ASPECTS STUDIED BY NMR .......................... 240
8.1 pH Titrations ......................................... 240
8.2 Ligand Binding ........................................ 241
8.2.1 Diffusion Studies .............................. 242
8.2.2 Mapping of the Binding Site .................... 242
8.2.3 Conformation of the Bound Ligand ............... 242
8.2.4 Changes in the Structure and Dynamics of the
Protein ........................................ 243
8.3 Enzyme Catalysis ...................................... 243
8.3.1 Dihydrofolate Reductase (DHFR) ................. 244
8.3.2 Triosephosphate Isomerase (TIM) ................ 244
8.4 Multi-Domain Structures ............................... 244
9 REFERENCES ................................................. 244
9.1 Historical Foundation of Protein Structure and
Function .............................................. 244
9.2 General Books on Proteins ............................. 245
9.3 Books on Protein NMR .................................. 245
9.4 Study of Large Proteins ............................... 245
9.5 Protein Structure, Dynamics and Folding ............... 246
9.6 Paramagnetic Proteins ................................. 246
9.7 Protein Binding and Function .......................... 246
CHAPTER 7: STRUCTURE AND DYNAMICS OF NUCLEIC ACIDS ............ 247
1 INTRODUCTION ............................................... 247
1.1 Chemical Constitution of Nucleic Acids ................ 247
1.2 Biological Role of Nucleic Acids ...................... 249
1.3 Historical Background of Nucleic Acid Structures ...... 250
2 ELEMENTS OF STRUCTURE OF NUCLEIC ACIDS ..................... 251
2.1 Nomenclature .......................................... 251
2.2 The Backbone Torsion Angles ........................... 253
2.3 Glycosidic Bond Rotation (χ) .......................... 253
2.4 Sugar Pucker: Pseudo-Rotation Angle (P) ............... 253
2.5 Inter-base Hydrogen Bonding ........................... 255
3 NMR SPECTROSCOPY OF NUCLEIC ACIDS .......................... 256
3.1 Comparison with Protein NMR ........................... 257
3.2 Classification of Chemical Shifts ..................... 258
3.3 Identification of Networks of Coupled Spin-systems .... 258
3.4 Sequence Specific Resonance Assignment Strategies ..... 261
3.5 Resonance Assignments using 13C and 15N Labelled
Nucleic Acids ......................................... 262
4 NMR PARAMETERS IN NUCLEIC ACIDS ............................ 264
4.1 Three Bond Coupling Constants (3J) .................... 264
4.2 31P-1H Couplings ....................................... 265
4.3 Scalar Couplings across Hydrogen Bonds ................ 265
4.4 Estimation of 1Н-1Н Distances using NOE ............... 266
4.5 Residual Dipolar Couplings (RDC) ...................... 266
4.6 Use of Paramagnetic Labels ............................ 267
5 STRUCTURE SIMULATIONS OF NUCLEIC ACIDS ..................... 267
5.1 Conformation of Deoxyribose and Ribose Rings .......... 268
5.2 Backbone Torsion Angles ............................... 271
5.3 Glycosidic Torsion Angle (χ) .......................... 271
5.4 Hydrogen-bond Constraints for Base-pairs .............. 271
5.5 3D Structure from NMR data ............................ 272
6 UNUSUAL DNA STRUCTURES ..................................... 273
6.1 Sequence Dependent Variations in B-DNA ................ 273
6.2 Mismatch Base-pairs ................................... 273
6.3 Hairpin Nucleic Acids ................................. 275
6.4 Parallel Stranded (ps) DNA Duplex ..................... 276
6.5 Triple Stranded Nucleic Acids ......................... 277
6.6 G-Quartet ............................................. 280
6.7 i-motif Tetraplex ..................................... 281
6.8 Cruciform Structure ................................... 282
6.9 Holliday or Four-way Junction ......................... 283
7 POLYMORPHISM IN DNA ........................................ 283
7.1 Concentration-induced Conformational Transition ....... 284
7.2 pH- Induced Polymorphism .............................. 286
7.3 Effect of Temperature ................................. 286
7.4 Effect of Metal Ions .................................. 287
8 DYNAMICS OF NUCLEIC ACIDS .................................. 287
9 REFERENCES ................................................. 288
9.1 Further Reading ....................................... 288
9.2 Books ................................................. 288
9.3 DNA Structure ......................................... 289
9.4 Parallel Stranded DNA ................................. 289
9.5 Triple-Helical Nucleic Acids .......................... 289
9.6 Isotope Labelling of Nucleic Acids .................... 290
CHAPTER 8: CARBOHYDRATES, LIPIDS AND LIPID ASSEMBLIES ......... 291
1 INTRODUCTION ............................................... 291
2 CARBOHYDRATES .............................................. 291
2.1 Biological Roles of Carbohydrates ..................... 292
2.2 Building Blocks of Carbohydrates ...................... 293
2.3 Oligosaccharides ...................................... 293
2.4 General Features of NMR of Carbohydrates .............. 294
2.5 Primary Structures of Carbohydrates ................... 297
2.6 NMR Determination of the Conformation of
Monosaccharides ....................................... 297
2.7 Assignments of NMR Spectrum of Carbohydrates .......... 298
2.8 Structure and Dynamics of Polysaccharides ............. 299
2.9 Glycoconjugates ....................................... 300
2.10 Applications in Development of Bacterial
Polysaccharides as Vaccines ........................... 301
2.11 Recognition Motifs in Polysaccharides ................. 302
2.12 Proteoglycans ......................................... 302
3 LIPIDS AND MODEL MEMBRANES ................................. 302
3.1 Elements of Biological Membranes ...................... 303
3.2 Lipids ................................................ 303
3.3 Conformation of Phospholipids ......................... 305
3.4 Lipid Vesicles and Bilayer Structures ................. 306
3.5 Membrane Architecture ................................. 307
4 NMR STUDIES OF STRUCTURE AND DYNAMICS OF LIPID BILAYERS .... 308
4.1 Molecular Motions in Model Membranes .................. 308
4.2 Phase Transitions ..................................... 309
4.3 Lipid Polymorphism in Model Membranes ................. 311
4.4 13C NMR Studies of Segmental Motions in Alkyl
Chains ................................................ 311
4.5 Molecular Order and 2H NMR Studies of Membrane
Organization .......................................... 312
4.6 Modulation of Motion and Order of Lipids by Larger
Molecules ............................................. 313
4.7 Measurement of Lateral Diffusion using NMR ............. 314
5 REFERENCES ................................................. 314
5.1 Further Reading ....................................... 314
5.2 Books and Reviews ..................................... 315
CHAPTER 9: HIGH-RESOLUTION SOLID-STATE NMR .................... 317
1 INTRODUCTION ............................................... 317
2 NMR HAMILTONIANS IN SOLID-STATE ............................ 317
2.1 NMR Hamiltonian in Solid-State ........................ 318
2.2 Chemical Shift Anisotropy (HCSA) ...................... 320
2.3 Heteronuclear Dipolar Interactions [Hhet(D)] ........... 323
2.4 Homonuclear Dipolar Interactions [Hhomo(D)] ............ 324
2.5 Quadrupolar Interaction (HQ) .......................... 325
2.6 Scalar Coupling [Hj] .................................. 326
3 STRATEGIES FOR OBTAINING HIGH-RESOLUTION NMR SPECTRA IN
SOLID-STATE ................................................ 326
3.1 Magic-Angle Spinning (MAS) ............................ 328
3.2 Heteronuclear Dipolar Decoupling (DD) ................. 329
3.3 Cross Polarization (CP) ............................... 330
3.4 31PNMR ................................................ 331
3.5 Spectra of Dilute Spin 1/2 Nuclei ..................... 331
3.6 1H NMR ................................................ 332
3.7 Heteronuclear and Homonuclear Correlation
Experiments ........................................... 333
3.8 2H NMR ................................................ 334
3.9 Measurement of Internuclear Distances in Solid-State:
Dipolar Recoupling .................................... 335
3.10 Reintroduction of CSA ................................. 336
4 PROTEIN STRUCTURE DETERMINATION USING SOLID-STATE NMR ...... 338
4.1 Membrane Proteins ..................................... 338
4.2 Polarisation Inversion at Magic Angle: PISA Wheels .... 340
4.3 Structure Analysis using MAS Solid-State NMR .......... 341
4.4 Ion Channel Proteins .................................. 341
4.5 Bacteriorhodopsin (bR) ................................ 342
4.6 Amyloid Proteins ...................................... 343
4.7 Structure of the Coat Protein in fd Filamentous
Bacteriophage Particles ............................... 345
4.8 Collagen .............................................. 345
4.9 Other Fibrous Proteins ................................ 347
4.10 Other Systems ......................................... 347
5 FUTURE OF SOLID-STATE NMR IN BIOLOGY ....................... 347
6 REFERENCES ................................................. 348
6.1 Further Reading ....................................... 348
6.2 Books and Reviews ..................................... 349
CHAPTER 10: BIOMOLECULAR INTERACTIONS AND SUPRAMOLECULAR
ASSEMBLIES ........................................ 351
1 INTRODUCTION ............................................... 351
2 INTERMOLECULAR INTERACTIONS ................................ 352
3 NMR APPROACHES TO STUDY BIOMOLECULAR INTERACTIONS .......... 352
3.1 Experimental Considerations ........................... 352
3.2 Use of NOE and RDC .................................... 353
3.3 Use of Perturbations in Chemical Shifts (CSPs) ........ 353
3.4 Relaxation Rates ...................................... 354
3.5 Use Paramagnetic Probes ............................... 354
3.6 Cross Saturation Method ............................... 355
3.7 Strategies for Structure Simulations .................. 356
4 PROTEIN-PROTEIN INTERACTIONS ............................... 356
5 PROTEIN-DNA INTERACTIONS ................................... 359
5.1 Biological Importance ................................. 359
5.2 Nature of Protein-Nucleic Acid Interactions ........... 360
5.3 Structure Calculation of Protein-Nucleic Acid
Complexes ............................................. 360
5.4 Examples of Protein-DNA Interactions .................. 361
5.4.1 Myocyte Enhancer Factor 2A (MEF2A)-DNA
Complex ........................................ 361
5.4.2 Far-upstream Element-Binding Protein (FBP)
Bound to Single-Stranded DNA ................... 363
6 RNA STRUCTURES AND THEIR INTERACTION WITH PROTEINS ......... 363
6.1 Structural Motifs in RNA Structures ................... 364
6.2 Introduction to NMR of RNA ............................ 364
6.3 Transfer RNA (tRNA) and its Interaction with
Aminoacyl-tRNA Synthetase ............................. 365
6.4 Ribosomal RNA ......................................... 368
6.5 Protein-RNA Interactions .............................. 369
6.6 NMR Studies of Protein-RNA Interactions ............... 369
6.6.1 JDV Tat-BIV TAR Complex ........................ 369
6.6.2 HIV-1 Rev peptide-RRE RNA complex .............. 370
6.6.3 Splicing Factor 1-RNA complex .................. 371
7 SUPRAMOLECULAR ASSEMBLIES .................................. 371
7.1 Bone .................................................. 371
7.2 Skeletal Muscle ....................................... 372
7.3 Large DNA and its Complexes ........................... 372
7.4 Chlorophyll ........................................... 372
8 FUTURE OF NMR STUDIES ON SUPRAMOLECULAR SYSTEMS ............ 374
9 REFERENCES ................................................. 374
9.1 Further Reading ....................................... 374
9.2 RNA Structure ......................................... 374
9.3 Protein-Nucleic acid Interactions ..................... 375
9.4 Books ................................................. 375
CHAPTER 11: MAGNETIC RESONANCE IMAGING ........................ 377
1 INTRODUCTION ............................................... 377
2 PRINCIPLES OF NMR IMAGING .................................. 377
3 SPATIAL LOCALIZATION OF THE NUCLEUS TO BE IMAGED ........... 379
3.1 Slice Selection ....................................... 380
3.2 Sensitive-Point Method ................................ 381
3.3 Two-Dimensional Fourier Imaging ....................... 382
3.3.1 Slice Selection ................................ 383
3.3.2 Phase Encoding ................................. 383
3.3.3 Frequency Encoding ............................. 384
3.3.4 Fourier Transform .............................. 384
3.3.5 Multi-slice Imaging ............................ 385
3.4 k-Space ............................................... 385
4 PULSE SEQUENCES IN MRI ..................................... 387
4.1 Spin-echo (SE) ........................................ 388
4.2 Half Fourier Transform Imaging (HFI) .................. 389
4.3 Inversion Recovery (IR) ............................... 390
4.4 The Need for Fast Imaging Techniques .................. 391
4.5 Gradient Echo (GRE) ................................... 392
4.6 Turbo-Spin-Echo (TSE) ................................. 393
4.7 Single-Shot Techniques: Echo Planar Imaging (EPI) ..... 395
4.8 Parallel Imaging ...................................... 396
4.9 Three Dimensional Imaging ............................. 397
4.10 Motion Suppression Techniques ......................... 397
4.11 Scan Speed ............................................ 397
4.12 Receiver Coils ........................................ 398
4.13 Display ............................................... 398
4.14 Future ................................................ 399
5 TISSUE CONTRAST BASED ON RELAXATION RATES AND 1H DENSITY ... 399
5.1 Proton Density (PD) ................................... 400
5.2 Relaxation times T1 and T2 ............................ 401
5.3 Achieving Contrast .................................... 402
5.4 Lipid Signals ......................................... 404
5.5 Susceptibility Effects: Diamagnetic Interfaces ........ 404
5.6 Paramagnetic and Ferromagnetic Materials: Contrast
Enhancement (CE) ...................................... 405
5.7 Other Imaging Parameters .............................. 406
6 IMAGE CONTRAST BASED ON FLOW, DIFFUSION AND PERFUSION ...... 406
6.1 Magnetic Resonance Angiography (MRA) .................. 407
6.2 Diffusion-weighted Imaging (DWI) ...................... 408
6.3 Perfusion ............................................. 410
6.4 Magnetization Transfer (MT) ........................... 411
7 FUNCTIONAL MRI (fMRI) ...................................... 411
7.1 Principle of fMRI ..................................... 411
7.2 Methodology ........................................... 412
7.3 Applications of fMRI .................................. 413
7.4 Limitations and Future Outlook ........................ 415
8 MRI AS A CLINICAL TOOL ..................................... 415
8.1 Central Nervous System (CNS) .......................... 415
8.1.1 Ischaemic Stroke ............................... 416
8.1.2 Haemorrhage Stroke ............................. 416
8.1.3 Neurological Diseases .......................... 417
8.2 Musculoskeletal System ................................ 417
8.3 Heart and Cardiovascular System ....................... 418
8.4 Breast ................................................ 418
8.5 MRI in Pregnancy ...................................... 420
8.6 Genitourinary System .................................. 420
8.7 Gastrointestinal System ............................... 420
8.8 Prostate Cancer ....................................... 420
8.9 Interventional Imaging ................................ 421
8.10 Patient Comfort and Precautions ....................... 421
9 REFERENCES ................................................. 422
9.1 Further Reading ....................................... 422
9.2 Books and Reviews ..................................... 422
CHAPTER 12: STUDY OF METABOLISM: CELLS AND TISSUES ............ 423
1 INTRODUCTION ............................................... 423
1.1 Cell-metabolism is the Bridge between Proteomics and
Function .............................................. 423
1.2 Measurement of Products of Metabolism ................. 424
2 METABOLIC CYCLES AND PATHWAYS .............................. 425
2.1 Glycolysis ............................................ 426
2.2 Citric Acid (Kreb's) Cycle ............................ 427
2.3 Oxidative Phosphorylation ............................. 428
2.4 Pentose Phosphate Pathway, Gluconeogenesis and
Glycogen Synthesis .................................... 428
2.5 Fatty Acid Synthesis and Degradation .................. 429
2.6 Amino Acid Metabolism ................................. 429
2.7 Integration and Control ............................... 429
2.8 Metabolic Profile and Requirements of each Body Organ
is Different .......................................... 430
2.9 31P NMR and Metabolism ................................ 431
2.10 1H NMR Spectroscopy ................................... 433
2.11 13C NMR Spectroscopy .................................. 434
3 STUDIES OF BODY FLUIDS ..................................... 435
3.1 Metabolomic Analysis using Biological Fluids .......... 435
3.2 1H and 13C Chemical Shifts of Common Metabolites:
Assignments ........................................... 437
3.3 Suppression of Water and Other Undesirable Signals .... 439
3.4 Multi-dimensional NMR and Quantification .............. 440
3.5 Multi-variant Statistical Analysis .................... 440
3.6 Applications of Metabonomics .......................... 441
4 CELLULAR NMR SPECTROSCOPY .................................. 442
4.1 Technical Aspects of NMR in Cells ..................... 443
4.2 Studies on Spermatozoa ................................ 444
4.3 Identification of Low Molecular Weight Compounds in
Cells ................................................. 444
4.4 Biochemical Changes during Cell Maturation,
Modification and Differentiation ...................... 445
4.5 Glycolysis in Cells ................................... 446
4.6 Effect of Exogenous Compounds on Metabolism ........... 447
4.7 In-Cell Studies of Macromolecular Structure and
Dynamics .............................................. 447
5 STUDIES OF TISSUES USING SOLID-STATE NMR TECHNIQUES ........ 449
6 REFERENCES ................................................. 451
6.1 Further Reading ....................................... 451
6.2 Books and Reviews ..................................... 452
CHAPTER 13: MRS STUDIES OF METABOLISM IN ANIMALS AND HUMANS ... 453
1 INTRODUCTION ............................................... 453
2 TECHNIQUES FOR DETECTING MR SIGNALS ........................ 454
2.1 Spectral Localization ................................. 454
2.2 Water Suppression ..................................... 456
2.3 Depth Resolved Surface Coil Spectroscopy (DRESS) ...... 456
2.4 Image Guided Protocols ................................ 457
2.5 Image Selected in vivo Spectroscopy (ISIS) ............ 457
2.6 Stimulated Echo Acquisition Mode Spectroscopy
(STEAM) ............................................... 458
2.7 Point Resolved Spectroscopy (PRESS) ................... 459
2.8 Comparison of STEAM and PRESS ......................... 459
2.9 Multiple Volume Spectroscopy (MVSI or CSI) ............ 460
2.10 Recent Developments in MRS ............................ 461
2.11 Detection of Metabolites using 2D NMR ................. 462
3 UNDERSTANDING THE CHEMISTRY OF BRAIN THROUGH MRS ........... 462
3.1 1H NMR Spectroscopy ................................... 463
3.2 13C NMR: The Glu, Gin, GABA Cycle ..................... 465
3.3 Age and Disease Related Changes in MRS of Brain ....... 466
4 MRS OF OTHER ORGANS ........................................ 466
4.1 Muscle ................................................ 467
4.2 Insulin Regulation of Glycogen Metabolism ............. 469
4.3 Breast ................................................ 469
4.4 Prostrate ............................................. 471
4.5 Cardiovascular Disorders .............................. 472
5 MR IMAGING AND SPECTOSCOPY USING OTHER NUCLEIAR SPINS ...... 472
5.1 Difficulties in vivo Spectroscopic Studies using
other Spins ........................................... 473
5.2 l9F ................................................... 473
5.3 2H .................................................... 475
5.4 7Li ................................................... 475
5.5 23Na .................................................. 476
5.6 3He and 129Xe ......................................... 476
5.7 Other Nuclear Spins ................................... 477
6 APPLICATIONS OF NMR IN DRUG DEVELOPMENT .................... 477
6.1 Stages in Drug Development ............................ 477
6.2 Target Validation and Receptor Identification ......... 478
6.3 Structural Approach to Drug-design .................... 479
6.4 Drug-Receptor Binding ................................. 480
6.5 Lead Identification: Ligands for Hot Spot ............. 480
6.6 Second Binding Site: The Linked-Fragment Strategy ..... 482
6.7 Lead Optimization ..................................... 482
6.8 Pre-clinical Studies: Drug Metabolism using Body
Fluids ................................................ 482
6.9 Human Trials .......................................... 483
6.10 Drug purity: Interfacing NMR with LC and MS ........... 484
6.11 Use of Solid-State NMR for Drug Powders ............... 484
7 REFERENCES ................................................. 485
7.1 Further Reading ....................................... 485
7.2 Books and Reviews ..................................... 486
CHAPTER 14: STRUCTURE AND METABOLISM OF PLANTS ................ 487
1 INTRODUCTION ............................................... 487
1.1 Cell Structure of Plants .............................. 487
1.2 Biochemistry of Plants ................................ 488
1.3 Importance of NMR in Plant Biochemistry ............... 488
2 UNIQUE METABOLIC PATHWAYS IN PLANTS ........................ 489
2.1 Photosynthesis ........................................ 489
2.2 Nitrogen Metabolism ................................... 489
2.3 Photorespiration ...................................... 489
2.4 Sulphur Metabolism .................................... 490
3 NMR OF PLANTS: GENERAL FEATURES ............................ 490
3.1 Handling Whole Plants ................................. 490
3.2 Tissues and Small Plants .............................. 492
3.3 General Features of Plant NMR ......................... 492
3.4 Scope of Plant NMR .................................... 493
3.5 Nuclear Spins used in Plant NMR ....................... 493
3.6 Interpretation of Spectra ............................. 495
3.7 Compartmentation in Plant Cells ....................... 495
4 PLANT METABOLISM USING NMR ................................. 496
4.1 Tissues, Cells and Cell Extracts ...................... 496
4.2 Nitrogen Metabolism ................................... 497
4.3 Amino Acid Metabolism ................................. 499
4.4 Photosynthesis ........................................ 499
4.5 Carbon Metabolism ..................................... 500
4.6 Sulphur Metabolism .................................... 500
4.7 Phosphorus Metabolism ................................. 501
4.8 Other ions and Substrates ............................. 501
4.9 Paramagnetic Reagents ................................. 501
4.10 Effect of Physiological Conditions .................... 502
4.11 Secondary Metabolic Pathways .......................... 502
4.12 Plant-Fungal Relations ................................ 502
5 METABOLIC FLUXES AND PLANT METABOLOMICS .................... 502
5.1 Analysis of Metabolic Fluxes .......................... 503
5.2 Secondary Products of Plant Metabolism ................ 503
5.3 Plant Metabolomics .................................... 503
6 IMAGING AND MICROSCOPY IN PLANTS ........................... 503
6.1 NMR Microscopy ........................................ 504
6.2 Studies of Plant Water ................................ 504
6.3 Studies of Roots ...................................... 505
6.4 Stems, Leaves and Flowers ............................. 505
6.5 Fruits and Seeds ...................................... 505
6.6 Chemical Shift Imaging ................................ 506
7 SOLID-STATE NMR STUDIES .................................... 506
7.1 Studies on Plant Organelles ........................... 506
7.2 Solid-State NMR on PS I and PS II ..................... 507
7.3 Chlorosomal Bacteriochlophylls ........................ 508
8 REFERENCES ................................................. 509
8.1 Further Reading ....................................... 509
8.2 Books and Reviews ..................................... 510
INDEX ......................................................... 511
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