Home Shop Service Jobs Newsletter Company Sitemap Entertainment Shopping cart Deutsch
Books | Chemistry | BioNMR in Drug Research | Table of contents
Browse our products
Books
 
Just published
Title search
Featured sites
Entertainment
Journals
Electronic Media
Choose your area of interest
 
  Contents  
 
  Preface XVII
  Foreword XIX
  List of Authors XXI
  List of Abbreviations XXVII
Part I Basic TechniquesContents  
1 Modern Methods for the Expression of Proteins in Isotopically Enriched Form
Heiko Patzelt, Natalie Goto, Hideo Iwai, Kenneth Lundstrom, and Erhard Fernholz 		1
1.1 Introduction 1
1.2 Isotope-Labeled Proteins from Hydrolyzates of the Green Alga Scenedesmus obliquus 12
1.2.1 Production of Isotope-Labeled Algal Hydrolyzates 3
1.2.2 Adaptation of the Protein Overproducer to the Algal Medium 4
1.2.3 Preparation of Homogenously Isotope-Labeled Protein by Fermentation on Algal Media 5
1.2.4 Amino Acid-Type Specific Labeling 5
1.2.5 Mass Spectrometric Analysis of the Labeled Amino Acids 6
1.3 Selective Labeling Schemes 6
1.3.1 Reverse-Labeling Schemes 8
1.3.1.1 Selective Protonation of Methyl Groups in 2H-Labeled Proteins 8
1.3.1.2 Structure Determination of Selectively Methyl Protonated Proteins 10
1.3.1.3 Introducting 1H,12C Aromatic Residues into Otherwise 13C Uniformly Labeled Proteins 10
1.3.1.4 Backbone-Labeled Proteins 10
1.3.2 Selective 13C Methyl Group Labeling 11
1.4 Intein-Based Protein Engineering for NMR Spectroscopy 11
1.4.1 Segmental Labeling of Proteins 13
1.4.1.1 Intein-Mediated Protein Ligation (IPL)/Expressed Protein Ligation (EPL) using the IMPACT System 13
1.4.1.2 Reconstitution of Split Inteins 15
1.4.2 Stabilizing Proteins by Intein-Mediated Backbone Cyclization 18
1.4.2.1 In vitro Cyclization of Proteins 18
1.4.2.2 In vivo Cyclization 20
1.4.2.3 Stability Enhancement by Backbone Cyclization 20
1.5 Alternatives to E. coli Expression Systems 20
1.5.1 Expression Vectors 21
1.5.1.1 Halobacterium salinarum 21
1.5.1.2 Saccharomyces cerevisiae 22
1.5.1.3 Schizosaccharomyces pombe 23
1.5.1.4 Pichia pastoris 23
1.5.1.5 Baculovirus 24
1.5.1.6 Transient Mammalian Expression 24
1.5.1.7 Stable Mammalian Expression 24
1.5.1.8 Viral Vectors 25
1.5.2 Comparison of Expression Systems 25
1.5.3 Isotope Labeling and NMR 27
1.5.4 Target Proteins 28
1.6 The Use of Cell-Free Protein Expression for NMR Analysis 29
1.6.1 The Cell-Free Protein Expression Systems RTS 30
1.6.2 From PCR Product to 15N-Labeled Protein 31
1.6.3 Discussion and Outlook 33
1.7 References 34
2 Structure Calculation Using Automated Techniques
Peter Güntert 		39
2.1 Introduction 39
2.2 Conformational Constraints for NMR Structure Calculations 39
2.2.1 Constraints from Covalent Structure 40
2.2.2 Steric Repulsion 40
2.2.3 Distance Constraints from Nuclear Overhauser Effects 40
2.2.4 Hydrogen Bond Distance Constraints 43
2.2.5 Torsion Angle Constraints from Chemical Shifts 43
2.2.6 Torsion Angle Constraints from Scalar Coupling Constants 44
2.2.7 Orientation Constraints 45
2.3 Structure Calculation Algorithms 46
2.3.1 Simulated Annealing by Molecular Dynamics Simulation in Cartesian Space 46
2.3.2 Torsion Angle Dynamics 48
2.4 Automated NOESY Assignment 52
2.4.1 The NOESY Assignment Problem 52
2.4.2 Semi-Automatic Methods 53
2.4.3 General Principles of Automatic NOESY Assignment 53
2.4.4 Requirements on Input Data 54
2.4.5 Overview of Algorithms 55
2.4.6 The Candid Algorithm 56
2.4.7 Network-Anchoring of NOE Assignments 59
2.4.8 Constraint-Combination 60
2.4.9 Has it worked? 62
2.5 References 64
3 Achieving Better Sensitivity, Less Noise and Fewer Artifacts in NMR Spectra
Detlef Moskau and Oliver Zerbe 		67
3.1 Introduction 67
3.2 The Transmitter and Receiver System 69
3.3 The Magnet, Shim and Lock System 71
3.4 Sample Conditions and Environmental Set-up 73
3.5 Probeheads 74
3.6 Acknowledgements 78
3.7 References 78
Part II NMR of Biomolecules  
4 NMR Strategies for Protein Assignments
Volker Dötsch 		79
4.1 Introduction 79
4.2 Optimization of Solution Conditions 79
4.3 Labeling and Overexpression 81
4.4 NMR Experiment 83
4.4.1 Small and Medium-Sized Proteins 83
4.4.2 Large Proteins 88
4.5 Assignment Procedures 90
4.6 References 92
5 NMR of Membrane-Associated Peptides and Proteins
Reto Bader, Mirjam Lerch, and Oliver Zerbe 		95
5.1 The Biochemistry of Membrane Interactions 95
5.1.1 Introduction 95
5.1.2 Biological Membranes 98
5.1.2.1 Protein-Membrane Interactions 99
5.1.3 Aggregate Structures of Lipids and their Biophysics 101
5.1.3.1 Micelles 101
5.1.3.2 Bicelles 103
5.1.3.3 Vesicles or Liposomes 103
5.2 The NMR Sample 104
5.2.1 Synthesis of Peptides and Proteins 104
5.2.2 Choice of Detergent 105
5.2.3 Choice of pH 106
5.2.4 Choice of Temperature 107
5.2.5 Salt Concentrations 108
5.2.6 Practical Tips for Sample Preparation 109
5.3 The Structure and Dynamics of Membrane-Associated Peptides -- A Case Study of Neuropeptide Y (NPY) 110
5.3.1 Introduction 110
5.3.2 Structure Determination of Micelle-Bound NPY 110
5.3.3 The Determination of the Topology of the Membrane-NPY Interface 112
5.3.3.1 Spin Labels 112
5.3.3.2 Amide H,D Exchange 114
5.3.4 Measurement of Internal Dynamics of NPY/DPC 114
5.4 References 117
6 NMR of Nucleic Acids
Radovan Fiala and Vladimír Sklená 		121
6.1 Introduction 121
6.2 Sample Preparation 122
6.3 Preparation of Labeled Nucleic Acids for Multinuclear NMR 123
6.4 Assignment Strategy -- New and Sensitivity-Optimized Experiments 124
6.5 NMR Detection of Hydrogen Bonds 131
6.6 Measurement of J-Couplings 134
6.7 Residual Dipolar Couplings -- Use for Structure Elucidation 134
6.8 Relaxation Studies of Nucleic Acids 138
6.9 Conclusions 143
6.10 Acknowledgements 144
6.11 References 144
Part III Modern Spectroscopic Techniques  
7 Methods for the Measurement of Angle Restraints from Scalar, Dipolar Couplings and from Cross-Correlated Relaxation: Application to Biomacromolecules
Christian Griesinger 		147
7.1 Introduction 147
7.2 Coupling Constants 147
7.2.1 The E. COSY Principle [8] 149
7.2.2 The DQ/ZQ Principle [9] 151
7.2.3 The FIDS Principle [13] 153
7.2.4 Quantitative J-correlation spectroscopy [15] 156
7.3 Incorporation of Dipolar Couplings into Simulated Annealing Protocols 159
7.4 Cross-Correlated Relaxation for the Measurement of Projection Angles between Tensors [18] 161
7.4.1 J-Resolved Constant Time Measurement of Cross-Correlated Relaxation Rates 165
7.4.2 Quantitative Measurement of Cross-Correlated Relaxation Rates 168
7.4.3 J-Resolved Constant Time Experiment for the Determination of the Phosphodiester Backbone Angles and 172
7.4.4 Transferred Cross-Correlated Relaxation 173
7.5 Applicability of Methods 174
7.6 References 176
8 Orientational Restraints
Eva de Alba and Nico Tjandra 		179
8.1 General Considerations 179
8.2 Commonly Used Systems to Orient Biopolymers 182
8.2.1 Bicelle Systems 182
8.2.2 Other Orienting Systems 183
8.3 NMR Experiments Designed to Measure Dipolar Couplings 184
8.4 Application of Dipolar Couplings to Structure Calculation 188
8.4.1 Protein Structure Determination and Refinement 188
8.4.2 Nucleic Acid Structure Calculation 192
8.4.3 Oligosaccharide Structure Calculation 193
8.5 Other Applications of Dipolar Couplings 197
8.5.1 Protein Structure Validation Factors 197
8.5.2 Protein Domain Orientation 198
8.5.3 Protein-Ligand Conformation and Orientation 198
8.5.4 Structure Building using Dipolar Couplings 199
8.5.5 Dipolar Couplings in Protein Family Search 201
8.6 Acknowledgements 202
8.7 References 202
9 Scalar Couplings Across Hydrogen Bonds
Andrew J. Dingley, Florence Cordier, Victor A. Jaravine, and Stephan Grzesiek 		207
9.1 Introduction 207
9.2 H-Bond Scalar Couplings in Biomacromolecules 210
9.2.1 Nucleic Acids 210
9.2.1.1 h2JNN-Couplings 210
9.2.1.2 h1JHN-Couplings 213
9.2.1.3 h3JNC-Couplings 213
9.2.2 Proteins 213
9.2.2.1 h3JNC-Couplings 213
9.2.2.2 h2JHC- and h3JHCa-Couplings 216
9.2.2.3 h2JNN-Couplings 216
9.2.2.4 h2JHMe-Couplings 216
9.2.3 Protein-Nucleic Acid Complexes 216
9.2.3.1 h2JNN-Couplings 216
9.2.3.2 h3JNP- and h2HP-Couplings 217
9.3 Relation to Chemical Shift 217
9.4 Dependence on Geometry 217
9.4.1 H-Bond Lengths 217
9.4.2 H-Bond Angles 218
9.5 Applications 219
9.5.1 Establishment of Secondary and Tertiary Sructure Information 219
9.5.2 Physicochemically-Induced Changes in H-Bond Geometry 220
9.5.3 Ligand-Induced Changes in H-Bond Geometry 220
9.5.4 Protein Folding 221
9.6 Conclusions 221
9.7 Acknowledgements 223
9.8 References 224
10 TROSY: Transverse Relaxation-Optimized Spectroscopy
Roland Riek 		227
10.1 Introduction 227
10.2 The Concept of TROSY 227
10.2.1 A Physical Picture of TROSY 228
10.2.2 Technical Aspects of TROSY 230
10.3 TROSY Applications 232
10.3.1 [15N,1H]-TROSY 232
10.3.2 [15N,1H]-TROSY -- Triple Resonance Spectroscopy for Sequential Assignment 233
10.3.3 [13C,1H]-TROSY 235
10.3.4 TROSY-Based NOESY Experiments 235
10.3.5 Transverse Relaxation-Optimization in the Polarization Transfers 235
10.4 Conclusions 236
10.5 Appendix: TROSY-Theory 237
10.6 Acknowledgements 240
10.7 References 240
11 MAS Solid-State NMR of Isotopically Enriched Biological Samples
Philip T.F. Williamson, Matthias Ernst, and Beat H. Meier 		243
11.1 Introduction 243
11.2 Basic Concepts in Solid-State NMR 244
11.2.1 Spin Interactions 244
11.2.1.1 The Chemical-Shift Hamiltonian 245
11.2.1.2 The Dipolar-Coupling Hamiltonian 246
11.2.1.3 The Quadrupolar Hamiltonian 247
11.2.1.4 The J-Coupling Hamiltonian 247
11.2.2 Basic Building Blocks for Solid-State NMR Experiments 248
11.2.2.1 Magic-Angle Spinning 248
11.2.2.2 Sensitivity-Enhancement Techniques 249
11.2.2.3 Heteronuclear Decoupling 250
11.3 Polarization-Transfer Techniques 252
11.3.1 Adiabatic Versus Sudden Polarization Transfer 252
11.3.2 Homonuclear Polarization Transfer 254
11.3.2.1 Dipolar Recoupling Techniques 254
11.3.2.2 J-Coupling Polarization-Transfer Techniques 258
11.3.3 Heteronuclear Polarization Transfer 259
11.3.3.1 Dipolar-Recoupling Techniques 259
11.3.3.2 J-Coupling Polarization-Transfer Techniques 261
11.3.4 A Comparison with Liquid-State NMR Methods 261
11.4 Experimental Considerations 262
11.4.1 Labeling Strategies 262
11.4.1.1 Specific Labeling Strategies for Small Peptides 262
11.4.1.2 Specific Labeling of Proteins 263
11.4.1.3 Chemical Labeling/Modification of Biomolecules 263
11.4.1.4 Uniform Labeling of Peptides and Proteins 264
11.4.1.5 Isotopic Dilution 264
11.4.2 Sample Preparation 266
11.4.2.1 Soluble Proteins 266
11.4.2.2 Membrane Proteins 266
11.5 Application of Polarization-Transfer Techniques to Biological Systems 267
11.5.1 Assignment of Resonances 267
11.5.2 Conformational Constraints 271
11.5.2.1 Homonuclear Distance Measurements 271
11.5.2.2 Heteronuclear Distance Measurements 273
11.5.2.3 Measurement of Torsion Angles 275
11.6 The Future of Applications/Developments of Solid-State NMR in Biology 277
11.7 References 277
12 Determination of Protein Dynamics Using 15N Relaxation Measurements
David Fushman 		283
12.1 Introduction 283
12.2 Spectroscopic Techniques 284
12.3 Accuracy and Precision of the Method 285
12.3.1 Sampling Schemes 285
12.3.2 Peak Integration 285
12.3.3 Estimation of Experimental Errors 285
12.3.4 Noise Reduction 286
12.3.5 Temperature Control 287
12.4 Basic Equations 288
12.5 The Model-Free Approach 289
12.6 Reduced Spectral Densities Mapping 290
12.7 Multi-Field Approach 291
12.8 Strategies for the Analysis of Protein Dynamics from 15N Relaxation Data 291
12.9 Overall Tumbling 292
12.10 How Can We Derive the Rotational Diffusion Tensor of a Molecule from Spin-Relaxation Data? 293
12.10.1 Theoretical Background 293
12.10.2 Derivation of the Diffusion Tensor when Protein Structure is Known 295
12.10.3 What Can We Do when Protein Structure is not Known? Preliminary Characterization of the Diffusion Tensor 296
12.10.4 Isotropic Overall Model 297
12.11 Model Selection for NH Bond Dynamics 298
12.12 Accuracy and Precision of the Model-Free Parameters 300
12.13 Motional Models 301
12.14 Conformational Exchange 301
12.15 Effects of Self-Association 303
12.16 Using 13C Relaxation to Study Protein Dynamics 304
12.17 What We Have Learned from Protein Dynamics Studies 305
12.18 Acknowledgements 305
12.19 References 306
Part IV Tools for Investigation of Drug -- Receptor Complexes and for Ligand Screening  
13 The Determination of Equilibrium Dissociation Constants of Protein-Ligand Complexes by NMR
Gordon C.K. Roberts 		309
13.1 Introduction 309
13.2 Chemical Exchange and NMR 309
13.3 The Basic Equations 312
13.4 Slow Exchange 314
13.5 Intermediate Exchange 314
13.6 Fast Exchange 314
13.6.1 Very Fast Exchange 315
13.6.2 The General Case of Fast Exchange 315
13.6.3 Paramagnetic Relaxation 317
13.7 Conclusions 317
13.8 References 319
14 Experiments in NMR-Based Screening
Carla Marchioro, Silvia Davalli, Stefano Provera, Markus Heller, Alfred Ross, and Hans Senn 		321
14.1 Introduction 321
14.2 NMR-Based Screening 323
14.2.1 Experiments Based on Chemical Shift Perturbations 325
14.2.2 Ligand-Observe Experiments 328
14.2.3 Experiments Based upon Changes in Relaxation Properties of Ligands 330
14.2.4 Diffusion-Editing Experiments 330
14.2.5 NOE-Based Techniques 335
14.2.6 Comparison of Methods 339
14.3 References 340
15 The Use of Spin Labels in NMR-Supported Lead Finding and Optimization
Wolfgang Jahnke 		341
15.1 Introduction 341
15.2 Basic Theory of Spin Labels 342
15.2.1 Some Practical Aspects of Work with Spin Labels 344
15.3 Applications of Spin Labels in NMR Screening 345
15.3.1 Primary NMR Screening Using Spin Labels: SLAPSTIC 345
15.3.2 Protein Amounts Needed for SLAPSTIC Screening 347
15.3.3 Validation and Preliminary Optimization of Primary NMR Screening Hits 349
15.3.4 Second-Site NMR Screening Using Spin Labels 350
15.4 Linker Design 352
15.5 Conclusions and Outlook 353
15.6 References 354
16 NMR of Weakly Binding Ligands
Marcel J.J. Blommers and Simon Rüdisser 		355
16.1 Introduction 355
16.2 The Dynamic Equilibrium 355
16.3 Transferred NOE (trNOE) 356
16.4 Transferred Cross-Correlated Relaxation (trCCR) 362
16.5 Transferred Residual Dipolar Couplings (trRDC) 367
16.6 Summary 369
16.7 References 369
17 Isotope Filter and Editing Techniques
Gerd Gemmecker 		373
17.1 General Concept 373
17.2 Sample Requirements 375
17.2.1 Complex Size and Concentration 375
17.2.2 Complex Affinity 376
17.2.3 Labeling Pattern 377
17.3 NMR Techniques 379
17.3.1 Heteronuclear Shift Correlations 379
17.3.2 Filtering and Editing Techniques 380
17.3.3 Selection of Intra-/Intermolecular NOEs 384
17.4 Applications 385
17.5 References 389
Part V Strategies for Drug Development Using NMR  
18 Strategies for NMR Screening and Library Design
Christopher A. Lepre 		391
18.1 Introduction 391
18.2 Choosing a Screening Strategy 391
18.2.1 Strategy Directs Library Design 391
18.2.2 Types of NMR Screening Strategies 392
18.2.3 Types of NMR Screening Libraries 396
18.3 Designing NMR Screening Libraries 399
18.3.1 Dealing with Diversity 399
18.3.2 Optimizing Molecular Complexity 401
18.3.3 Selecting for Drug-like Character 403
18.3.4 Solubility Requirements 404
18.3.5 Designing Mixtures 405
18.4 Implementing a Strategy 406
18.4.1 Choosing an Experimental Method 406
18.4.2 NMR Screening at Vertex 407
18.5 Conclusion 410
18.6 Acknowledgements 411
18.7 References 411
19 Strategies for Hit Finding Using NMR
Werner Klaus and Hans Senn 		417
19.1 Introduction 417
19.2 Hit Finding by NMR: the ´´Needle Concept´´ for de novo Screening 418
19.3 Requirements for NMR-based Screening 419
19.3.1 The Sample 419
19.3.2 The Ligands 420
19.3.4 Automation of NMR Experiments and Hardware Improvements 421
19.3.5 Automation in Spectral Analysis 423
19.4 Examples 424
19.4.1 Gyrase 424
19.4.2 Peptide Deformylase 427
19.4.3 MMP-1 430
19.5 Summary 433
19.6 Acknowledgements 436
19.7 References 436
20 Strategies for Drug Discovery Using NMR
Marcel J.J. Blommers, Andreas Flörsheimer, and Wolfgang Jahnke 		439
20.1 Introduction 439
20.1.1 Many Drugs are Modular 439
20.1.2 Fragments can be Assembled Piece by Piece 440
20.1.3 Three Alternative Approaches for the Discovery of Linked Fragments: In Silico Screening, SAR-by-NMR and Second-Site Screening 441
20.1.4 Hit Validation by NMR 442
20.2 Detection of Ligand Binding by NMR for Hit Validation of NMR Screening 443
20.2.1 Change in Relaxation Properties 443
20.2.2 Changes in Chemical Shift 443
20.2.3 Saturation Transfer 445
20.2.4 Transferred NOE 447
20.3 ´´Free Check´´ for Compound Solubility and Integrity 447
20.3.1 Solubility 447
20.3.2 Compound Integrity 449
20.4 Spin Labeling for Second-Site Screening 450
20.5 Intermolecular trNOE for Linker Design 451
20.6 A Case Study: Application of Second-Site Screening to Tubulin 451
20.7 An Integrated NMR Approach 455
20.8 References 457
21 NMR-Based Drug Design: Approaches for Very Large Proteins
Maurizio Pellecchia, Xuemei Huang, David Meininger, and Daniel S. Sem 		459
21.1 Introduction 459
21.2 NMR with Very Large Proteins 459
21.2.1 Protein Perdeuteration and SEA-TROSY (Solvent Exposed Amides with Transverse Relaxation Optimized Spectroscopy) 460
21.2.2 Protein Perdeuteration and Selective Amino Acid Labeling 464
21.3 NMR-Based Drug Design Techniques 465
21.3.1 NMR-DOC (Nuclear Magnetic Resonance Docking of Compounds) 465
21.3.2 NMR-SOLVE (Nuclear Magnetic Resonance Structurally Oriented Library Valency Engineering) 469
21.4 Conclusions 470
21.5 References 471
  Subject Index 473

 
Order
Online book
Table of contents
Short description
Detailed description
Reviews
Related volumes in series
Author affiliation
Bookmark and Share

Related Books

Classics in Spectroscopy
Isolation and Structure Elucidation of Natural Products

Classics in Spectroscopy
Isolation and Structure Elucidation of Natural Products

Ein- und zweidimensionale NMR-Spektroskopie
Eine Einführung


[more >>]

Related Journals

Angewandte Chemie

Angewandte Chemie International Edition

Chemie Ingenieur Technik


[more>>]

Special Offers

Ballwieser, Wolfgang / Beine, Frank / Hayn, Sven / Peemöller, Volker H. / Schruff, Lothar / Weber, Claus-Peter (eds.)

Handbuch IFRS 2010
99.90 Euro
valid until
30 April 2010

[more offers >>]


 

Tell a friend          RSS Feeds             Print-Version

©2010 Wiley-VCH Verlag GmbH & Co. KGaA - Provider
http://www.wiley-vch.de - mailto: info@wiley-vch.de
Data Protection