Biomolecular and Bioanalytical Techniques

List of Contributors xix Preface xxiii 1 Principles of Health and Safety and Good Laboratory Practice 1 Elaine Armstrong 1.1 Introduction 1 1.2 Good Laboratory Practice 1 1.3 Risk Assessment 2 1.4 Chemical Risk Assessment 5 1.5 Biological Materials and Genetically Modified Organisms 7 1.6 Vacuum Apparatus, Pressure Systems and Associated Glassware 9 1.7 Cryogenic Liquefied Gases 9 1.8 Compressed Gas Cylinders 10 1.9 Electromagnetic Radiation 10 1.10 Lasers 11 1.11 High Magnetic Fields 11 1.12 Sharps 12 1.13 Ergonomic Issues 12 References 15 2 Applications of Chemoinformatics in Drug Discovery 17 Valerie J. Gillet 2.1 Significance and Background 17 2.2 Computer Representation of Chemical Structures 17 2.3 Database Searching 18 2.4 Practical Issues on Representation 22 2.5 Virtual Screening 22 2.6 Ligand-Based Virtual Screening 23 2.7 Protein-Ligand Docking 30 2.8 Evaluating Virtual Screening Methods 32 2.9 Case Studies of Virtual Screening 33 2.10 Conclusions 34 References 34 Further Reading 36 3 Bioinformatics and Its Applications in Genomics 37 David J. Parry-Smith 3.1 Significance and Short Background 37 3.2 Theory/Principles 40 3.3 Databases 43 3.4 Techniques 45 3.5 Applications 53 3.6 Concluding Remarks 56 References 56 Further Reading 57 Websites 57 4 Gene Cloning for the Analysis of Gene Expression 59 Huw B. Thomas and Raymond T. O'Keefe 4.1 Identifying Target Sequence 59 4.2 In Silico Design 59 4.3 Primer Design 60 4.4 Template Preparation 62 4.5 Cloning Methods 64 4.6 Uses for Cloned DNA Sequences 66 4.7 Verifying Cloned Sequences 67 4.8 Applications of Gene Constructs 68 4.9 Case Study: Cloning of a Human Missense Variant Exon into a Minigene Splicing Vector 69 4.10 Case Study: Epitope Tagging of a Yeast Gene 74 References 79 Further Reading 79 Websites 80 5 Proteomic Techniques and Their Applications 81 Hsueh-Fen Juan 5.1 Significance and Background 81 5.2 Principles of Major Proteomics Techniques 82 5.3 Methods for Proteomics 85 5.4 Applications 92 5.5 Concluding Remarks 95 Acknowledgements 95 References 95 Further Reading 99 Website Resources 99 6 Overproduction, Separation and Purification of Affinity-Tagged Proteins from Escherichia coli 101 Finbarr Hayes and Daniela Barillà 6.1 Introduction 101 6.2 Selecting an Affinity Tag: Glutathione-S-Transferase, Maltose-Binding Protein and Hexa-Histidine Motifs 103 6.3 The pET Vector Series: Archetypal Expression Vectors in E. coli 105 6.4 IMAC of a His6-Tagged Protein: Example Methodology with the ParF DNA Segregation Protein 107 6.5 Production and Purification of a GST-Tagged Protein: Example Methodology with the C-Terminal Domain of Yeast RNA Polymerase II 112 6.6 Further Purification of Tagged Proteins 114 6.7 Alternative Hosts for Protein Production 116 6.8 Concluding Remarks 116 Acknowledgements 117 References 117 Further Reading 121 7 Chromatography: Separation Techniques in Biology 123 W John Lough and Mark Carlile 7.1 Introduction to Chromatographic Separation 123 7.2 General Considerations for Protein Separation by Chromatography 132 7.3 Engineering Proteins for Streamlined Chromatographic Separations 142 7.4 Example Chromatographic Separations of Biological Samples 143 7.5 Other Applications of Chromatography for Biological Sample Preparation and Analysis 147 References 147 Further Reading 152 8 Synthetic Methodology in Chemical Biology 153 Richard C. Brewster and Stephen Wallace 8.1 Introduction 153 8.2 Peptide Synthesis 153 8.3 Amide Bond Synthesis 154 8.4 Bioorthogonal Chemistry 164 8.5 The Copper-Catalysed Azide-Alkyne Cycloaddition Reaction (CuAAC) 165 8.6 Unnatural Amino Acid Incorporation 169 8.7 Case Studies 172 8.8 Conclusion 176 References 177 Further Reading 178 9 Reaction Chemical Kinetics in Biology 179 Nicholas J. Harmer and Mirella Vivoli Vega 9.1 Significance 179 9.2 Overview of Kinetics and Its Application to Biology 180 9.3 Determination of Enzyme Kinetic Mechanisms 188 9.4 Technique/Protocol: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme and Use of Product Inhibition to Determine Mechanism 201 9.5 Case Study: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme 210 9.6 More Advanced Methods 212 9.7 Concluding Remarks 214 References 214 10 Mass Spectrometry and Its Applications 219 Blagojce Jovcevski and Tara L. Pukala 10.1 Significance 219 10.2 Theories and Principles of Biomolecular Mass Spectrometry 221 10.3 Techniques and Methodology in Biomolecular Mass Spectrometry 234 10.4 Applications 242 10.5 Concluding Remarks 248 Abbreviations 248 References 249 Further Reading 253 11 Applications and Complementarity of Analytical Ultracentrifugation and Light-Scattering Techniques 255 Chad A. Brautigam 11.1 Introduction 255 11.2 Analytical Ultracentrifugation 255 11.3 Light Scattering 262 11.4 Protocols 266 11.5 Applications 270 11.6 Conclusions 275 Acknowledgements 275 References 276 Further Reading 278 12 Application of Isothermal Titration Calorimetry (ITC) to Biomolecular Interactions 279 Graeme L. Conn 12.1 Introduction 279 12.2 Principles and Theory of ITC 281 12.3 Protocols for Design, Implementation and Analysis of ITC Experiments 285 12.4 Example Applications of ITC to Analysis of Biomolecular Interactions 299 12.5 Concluding Remarks 304 Acknowledgements 304 References 304 Further Reading 305 Website Resources 305 13 An Introduction to Infra-red and Raman Spectroscopies for Pharmaceutical and Biomedical Studies 307 Ka Lung Andrew Chan 13.1 Significance and Short Background 307 13.2 Theory 307 13.3 Technique/Methodology/Protocol 313 13.4 Applications 323 13.5 Concluding Remarks 327 References 327 Further Reading 331 14 Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes 333 Nathan N. Alder 14.1 Significance and Background 333 14.2 Theory and Principles 334 14.3 Techniques, Methodologies and Protocols 341 14.4 Case Studies: Fluorescence Spectroscopy to Analyse Membrane Protein Structural Dynamics 358 14.5 Concluding Remarks 360 Acknowledgements 361 References 361 Further Reading 363 15 Circular Dichroism and Related Spectroscopic Techniques 365 Sophia C. Goodchild, Krishanthi Jayasundera and Alison Rodger 15.1 Significance and Background 365 15.2 Theory/Principles 366 15.3 Technique/Methodology/Protocol 371 15.4 Applications 376 15.5 Concluding Remarks 382 References 382 Further Reading 384 16 Principles and Practice in Macromolecular X-Ray Crystallography 385 Arnaud Baslé and Richard J. Lewis 16.1 Significance and Short Background 385 16.2 Theory and Principles: Overview 385 16.3 Methodology 397 16.4 Applications 412 16.5 Concluding Remarks 414 Acknowledgements 414 References 414 Further Reading 418 17 Biomolecular NMR Spectroscopy and Structure Determination of DNA 421 Tony Cheung and Vasudevan Ramesh 17.1 Significance and Background 421 17.2 Basic NMR Theory 422 17.3 Multidimensional NMR Spectroscopy 428 17.4 NMR Instrumentation and Experiments 429 17.5 Structure and Conformational Parameters of DNA 435 17.6 NMR Structure Determination 440 17.7 Case Study 1: NMR Structure Determination and Conformational Analysis of 17mer Canonical GC DNA 446 17.8 Case Study 2: NMR Structure Determination and Conformational Analysis of 13mer 6-ThioguanineModified GC DNA 460 17.9 Conclusion 464 References 466 Further Reading 469 18 Cryo-TEM and Biological Structure Determination 471 SzymonW. Manka and Carolyn A. Moores 18.1 Significance and Background 471 18.2 Theoretical Principles of Biological Cryo-TEM 473 18.3 Experimental Approaches in Biological Cryo-TEM 481 18.4 Cryo-TEM Case Studies 491 18.5 Concluding Remarks 496 Acknowledgements 496 References 496 Website Resources 499 19 Computer Modelling and Molecular Dynamics Simulation of Biomolecules 501 Maria Reif and Martin Zacharias 19.1 Significance 501 19.2 Theory and Principles 502 19.3 Methodology 506 19.4 Applications 525 19.5 Concluding Remarks 528 References 529 Further Reading 535 Index 537