Wilson & Walker 8th Edition.pdf

Wilson and Walker’s Principles and Techniques of BIOCHEMISTRY AND MOLECULAR BIOLOGY Bringing this best-selling textbook right up-to-date, the new edition uniquely integrates the theories and methods that drive the fields of biology, biotechnology and medicine, comprehensively covering both the te...

Wilson and Walker’s Principles and Techniques of BIOCHEMISTRY AND MOLECULAR BIOLOGY Bringing this best-selling textbook right up-to-date, the new edition uniquely integrates the theories and methods that drive the fields of biology, biotechnology and medicine, comprehensively covering both the techniques students will encounter in lab classes and those that underpin current key advances and discoveries. The contents have been updated to include both traditional and cutting- edge techniques most commonly used in current life science research. Emphasis is placed on understanding the theory behind the techniques, as well as analysis of the resulting data. New chapters cover proteomics, genomics, metabolomics and bioinformatics, as well as data analysis and visualisation. Using accessible language to describe concepts and methods, and with a wealth of new in-text worked examples to challenge students’ understanding, this textbook provides an essential guide to the key techniques used in current bioscience research. ASSOCIATE PROFESSOR ANDREAS HOFMANN is the Structural Chemistry Program Leader at Griffith University’s Eskitis Institute and an Honorary Senior Research Fellow at the Faculty of Veterinary and Agricultural Sciences at The University of Melbourne. He is also Fellow of the Higher Education Academy (UK). Professor Hofmann’s research lab focusses on the structure and function of proteins in infectious and neurodegenerative diseases, and also develops computational tools. He is the author of Methods of Molecular Analysis in the Life Sciences (2014) and Essential Physical Chemistry (2018). DR SAMUEL CLOKIE is the Principal Clinical Bioinformatician at the Birmingham Women’s and Children’s Hospital, UK, where he leads the implementation of bioinformatic techniques to decipher next-generation sequencing data in clinical diagnostics. He is also Honorary Senior Research Fellow at the Institute of Cancer and Genomic Sciences at The University of Birmingham and before that was a research fellow at the National Institutes of Health in Bethesda, USA. Wilson and Walker’s Principles and Techniques of Biochemistry and Molecular Biology Edited by ANDR EAS HOF MANN Griffith University, Queensland SAM UEL CL O KIE Birmingham Women’s and Children’s Hospital University Printing House, Cambridge CB2 8BS, United Kingdom One Liberty Plaza, 20th Floor, New York, NY 10006, USA 477 Williamstown Road, Port Melbourne, VIC 3207, Australia 314–321, 3rd Floor, Plot 3, Splendor Forum, Jasola District Centre, New Delhi – 110025, India 79 Anson Road, #06-04/06, Singapore 079906 Cambridge University Press is part of the University of Cambridge. It furthers the University’s mission by disseminating knowledge in the pursuit of education, learning, and research at the highest international levels of excellence. www.cambridge.org Information on this title: www.cambridge.org/9781107162273 DOI: 10.1017/9781316677056 © Andreas Hofmann and Samuel Clokie 2018 This publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 2018 Printed in the United Kingdom by Clays, St Ives plc A catalogue record for this publication is available from the British Library. Library of Congress Cataloging-in-Publication Data Names: Wilson, Keith, 1936– editor. | Walker, John M., 1948– editor. | Hofmann, Andreas, editor. | Clokie, Samuel, editor. Title: Wilson and Walker's principles and techniques of biochemistry and molecular biology / edited by Andreas Hofmann, Griffith University, Queensland, Samuel Clokie, Birmingham Women's and Children's Hospital. Other titles: Principles and techniques of biochemistry and molecular biology. Description: [2018 edition]. | Cambridge : Cambridge University Press, 2018. | Includes bibliographical references and index. Identifiers: LCCN 2017033452 | ISBN 9781107162273 (alk. paper) Subjects: LCSH: Biochemistry – Textbooks. | Molecular biology – Textbooks. Classification: LCC QP519.7.P75 2018 | DDC 572.8–dc23 LC record available at https://lccn.loc.gov/2017033452 ISBN 978-1-107-16227-3 Hardback ISBN 978-1-316-61476-1 Paperback Additional resources for this publication at www.cambridge.org/hofmann Cambridge University Press has no responsibility for the persistence or accuracy of URLs for external or third-party internet websites referred to in this publication and does not guarantee that any content on such websites is, or will remain, accurate or appropriate.  CONTENTS Foreword to the Eighth Edition page xiii Preface xv List of Contributors xvii 0 Tables and Resources xxi 1 Biochemical and Molecular Biological Methods in Life Sciences Studies 1 Samuel Clokie and Andreas Hofmann 1.1 From Biochemistry and Molecular Biology to the Life Sciences 1 1.2 The Education of Life Scientists 2 1.3 Aims of Life Science Studies 3 1.4 Personal Qualities and Scientific Conduct 5 1.5 Suggestions for Further Reading 7 2 Basic Principles 8 Parisa Amani and Andreas Hofmann 2.1 Biologically Important Molecules 8 2.2 The Importance of Structure 9 2.3 Parameters of Biological Samples 18 2.4 Measurement of the pH: The pH Electrode 24 2.5 Buffers 26 2.6 Ionisation Properties of Amino Acids 30 2.7 Quantitative Biochemical Measurements 32 2.8 Experiment Design and Research Conduct 33 2.9 Suggestions for Further Reading 38 3 Cell Culture Techniques 40 Anwar R. Baydoun 3.1 Introduction 40 3.2 The Cell Culture Laboratory and Equipment 41 3.3 Safety Considerations in Cell Culture 46 3.4 Aseptic Techniques and Good Cell Culture Practice 46 3.5 Types of Animal Cells, Characteristics and Maintenance in Culture 51 3.6 Stem Cell Culture 61 3.7 Bacterial Cell Culture 67 3.8 Potential Use of Cell Cultures 70 v vi Contents 3.9 Acknowledgements 71 3.10 Suggestions for Further Reading 71 4 Recombinant DNA Techniques and Molecular Cloning 73 Ralph Rapley 4.1 Introduction 73 4.2 Structure of Nucleic Acids 74 4.3 Genes and Genome Complexity 80 4.4 Location and Packaging of Nucleic Acids 83 4.5 Functions of Nucleic Acids 85 4.6 The Manipulation of Nucleic Acids: Basic Tools and Techniques 96 4.7 Isolation and Separation of Nucleic Acids 98 4.8 Automated Analysis of Nucleic Acid Fragments 103 4.9 Molecular Analysis of Nucleic Acid Sequences 104 4.10 The Polymerase Chain Reaction (PCR) 110 4.11 Constructing Gene Libraries 118 4.12 Cloning Vectors 128 4.13 Hybridisation and Gene Probes 145 4.14 Screening Gene Libraries 146 4.15 Applications of Gene Cloning 148 4.16 Expression of Foreign Genes 153 4.17 Analysing Genes and Gene Expression 157 4.18 Analysing Genetic Mutations and Polymorphisms 167 4.19 Molecular Biotechnology and Applications 173 4.20 Pharmacogenomics 175 4.21 Suggestions for Further Reading 176 5 Preparative Protein Biochemistry 179 Samuel Clokie 5.1 Introduction 179 5.2 Determination of Protein Concentrations 180 5.3 Engineering Proteins for Purification 184 5.4 Producing Recombinant Protein 188 5.5 Cell-Disruption Methods 191 5.6 Preliminary Purification Steps 195 5.7 Principles of Liquid Chromatography 196 5.8 Chromatographic Methods for Protein Purification 206 5.9 Other Methods of Protein Purification 210 5.10 Monitoring Protein Purification 213 5.11 Storage 217 5.12 Suggestions for Further Reading 218 6 Electrophoretic Techniques 219 Ralph Rapley 6.1 General Principles 219  Contents vii 6.2 Support Media and Buffers 223 6.3 Electrophoresis of Proteins 226 6.4 Electrophoresis of Nucleic Acids 240 6.5 Capillary Electrophoresis 246 6.6 Microchip Electrophoresis 250 6.7 Suggestions for Further Reading 252 7 Immunochemical Techniques 253 Katja Fischer 7.1 Introduction 253 7.2 Antibody Preparation 261 7.3 Immunoassay Formats 271 7.4 Immuno Microscopy 277 7.5 Lateral Flow Devices 278 7.6 Epitope Mapping 279 7.7 Immunoblotting 279 7.8 Fluorescence-Activated Cell Sorting (FACS) 280 7.9 Cell and Tissue Staining Techniques 281 7.10 Immunocapture Polymerase Chain Reaction (PCR) 281 7.11 Immunoaffinity Chromatography 282 7.12 Antibody-Based Biosensors 282 7.13 Luminex® Technology 283 7.14 Therapeutic Antibodies 283 7.15 Suggestions for Further Reading 285 8 Flow Cytometry 287 John Grainger and Joanne Konkel 8.1 Introduction 287 8.2 Instrumentation 289 8.3 Fluorescence-Activated Cell Sorting (FACS) 294 8.4 Fluorescence Labels 294 8.5 Practical Considerations 298 8.6 Applications 305 8.7 Suggestions for Further Reading 312 9 Radioisotope Techniques 313 Robert J. Slater 9.1 Why Use a Radioisotope? 313 9.2 The Nature of Radioactivity 314 9.3 Detection and Measurement of Radioactivity 322 9.4 Other Practical Aspects of Counting Radioactivity and Analysis of Data 334 9.5 Safety Aspects 340 9.6 Suggestions for Further Reading 344 viii Contents 10 Principles of Clinical Biochemistry 346 Gill Rumsby 10.1 Principles of Clinical Biochemical Analysis 346 10.2 Clinical Measurements and Quality Control 350 10.3 Examples of Biochemical Aids to Clinical Diagnosis 361 10.4 Suggestions for Further Reading 380 11 Microscopy 381 Stephen W. Paddock 11.1 Introduction 381 11.2 The Light Microscope 384 11.3 Optical Sectioning 396 11.4 Imaging Live Cells and Tissues 403 11.5 Measuring Cellular Dynamics 407 11.6 The Electron Microscope 411 11.7 Image Management 417 11.8 Suggestions for Further Reading 420 12 Centrifugation and Ultracentrifugation 424 Kay Ohlendieck and Stephen E. Harding 12.1 Introduction 424 12.2 Basic Principles of Sedimentation 425 12.3 Types, Care and Safety Aspects of Centrifuges 430 12.4 Preparative Centrifugation 438 12.5 Analytical Ultracentrifugation 446 12.6 Suggestions for Further Reading 452 13 Spectroscopic Techniques 454 Anne Simon and Andreas Hofmann 13.1 Introduction 454 13.2 Ultraviolet and Visible Light Spectroscopy 460 13.3 Circular Dichroism Spectroscopy 471 13.4 Infrared and Raman Spectroscopy 476 13.5 Fluorescence Spectroscopy 479 13.6 Luminometry 492 13.7 Atomic Spectroscopy 494 13.8 Rapid Mixing Techniques for Kinetics 497 13.9 Suggestions for Further Reading 498 14 Basic Techniques Probing Molecular Structure and Interactions 500 Anne Simon and Joanne Macdonald 14.1 Introduction 500 14.2 Isothermal Titration Calorimetry 501 14.3 Techniques to Investigate the Three-Dimensional Structure 502 14.4 Switch Techniques 521  Contents ix 14.5 Solid-Phase Binding Techniques with Washing Steps 524 14.6 Solid-Phase Binding Techniques Combined with Flow 526 14.7 Suggestions for Further Reading 532 15 Mass Spectrometric Techniques 535 Sonja Hess and James I. MacRae 15.1 Introduction 535 15.2 Ionisation 537 15.3 Mass Analysers 549 15.4 Detectors 556 15.5 Other Components 557 15.6 Suggestions for Further Reading 557 16 Fundamentals of Bioinformatics 559 Cinzia Cantacessi and Anna V. Protasio 16.1 Introduction 559 16.2 Biological Databases 560 16.3 Biological Data Formats 563 16.4 Sequence Alignment and Tools 569 16.5 Annotation of Predicted Peptides 576 16.6 Principles of Phylogenetics 584 16.7 Suggestions for Further Reading 596 17 Fundamentals of Chemoinformatics 599 Paul Taylor 17.1 Introduction 599 17.2 Computer Representations of Chemical Structure 602 17.3 Calculation of Compound Properties 614 17.4 Molecular Mechanics 616 17.5 Databases 626 17.6 Suggestions for Further Reading 627 18 The Python Programming Language 631 Tim J. Stevens 18.1 Introduction 631 18.2 Getting Started 632 18.3 Examples 661 18.4 Suggestions for Further Reading 676 19 Data Analysis 677 Jean-Baptiste Cazier 19.1 Introduction 677 19.2 Data Representations 679 19.3 Data Analysis 702 19.4 Conclusion 730 19.5 Suggestions for Further Reading 730 x Contents 20 Fundamentals of Genome Sequencing and Annotation 732 Pasi K. Korhonen and Robin B. Gasser 20.1 Introduction 732 20.2 Genomic Sequencing 733 20.3 Assembly of Genomic Information 737 20.4 Prediction of Genes 742 20.5 Functional Annotation 745 20.6 Post-Genomic Analyses 752 20.7 Factors Affecting the Sequencing, Annotation and Assembly of Eukaryotic Genomes, and Subsequent Analyses 754 20.8 Concluding Remarks 755 20.9 Suggestions For Further Reading 755 21 Fundamentals of Proteomics 758 Sonja Hess and Michael Weiss 21.1 Introduction: From Edman Sequencing to Mass Spectrometry 758 21.2 Digestion 759 21.3 Tandem Mass Spectrometry 759 21.4 The Importance of Isotopes for Finding the Charge State of a Peptide 765 21.5 Sample Preparation and Handling 767 21.6 Post-Translational Modification of Proteins 769 21.7 Analysing Protein Complexes 772 21.8 Computing and Database Analysis 776 21.9 Suggestions for Further Reading 777 22 Fundamentals of Metabolomics 779 James I. MacRae 22.1 Introduction: What is Metabolomics? 779 22.2 Sample Preparation 780 22.3 Data Acquisition 785 22.4 Untargeted and Targeted Metabolomics 787 22.5 Chemometrics and Data Analysis 797 22.6 Further Metabolomics Techniques and Terminology 802 22.7 Suggestions for Further Reading 807 23 Enzymes and Receptors 809 Megan Cross and Andreas Hofmann 23.1 Definition and Classification of Enzymes 809 23.2 Enzyme Kinetics 813 23.3 Analytical Methods to Investigate Enzyme Kinetics 831 23.4 Molecular Mechanisms of Enzymes 838 23.5 Regulation of Enzyme Activity 840 23.6 Receptors 844 23.7 Characterisation of Receptor–Ligand Binding 845 23.8 Suggestions for Further Reading 862  Contents xi 24 Drug Discovery and Development 864 David Camp 24.1 Introduction 864 24.2 Molecular Libraries and Drug-Discovery Strategies 865 24.3 Assembling a Molecular Library 881 24.4 Compound Management 887 24.5 Screening Strategies Used in Hit Discovery 889 24.6 Active-to-Hit Phase 895 24.7 Hit-to-Lead Phase 895 24.8 ADMET 896 24.9 Lead Optimisation 903 24.10 Suggestions for Further Reading 903 Index 906 FOREWORD TO THE EIGHTH EDITION When the first edition of our book was published in 1975, biochemistry was emerging as a new discipline that had the potential to unify the previously divergent -ologies that constituted the biological sciences. It placed emphasis on the understanding of the structure, function and expression of individual proteins and their related genes, and relied on the application of analytical techniques such as electrophoresis, chro- matography and various forms of spectrometry. In the ensuing 40 years the com- pletion of the Human Genome Project has confirmed the central role of DNA in all of the activities of individual cells and the emergence of molecular biology as the means of understanding complex biological processes. This in turn has led to the new disciplines of bioinformatics, chemoinformatics, proteomics and metabolomics. The succeeding six editions of our book have attempted to reflect this evolution of biochemistry as a unifying discipline. All editions have placed emphasis on the exper- imental techniques that undergraduates can expect to encounter during the course of their university studies and to this end we have been grateful for the excellent feed- back we have received from the users of the book. The point has now been reached where we believed that it was appropriate for us to hand over the direction and academic balance of future editions to a new editorial team and to this end we are delighted that Andreas Hofmann and Samuel Clokie have agreed to take on the role. We wish them well and look forward to the continuing success of the book. K E IT H WILS O N A N D JO H N WA LK E R xiii PREFACE It has been a tremendous honour being asked by Keith Wilson, John Walker and Cambridge University Press to take on the role of editorship for this eighth edition of Principles and Techniques. In designing the content, we extend the long and successful tradition of this text to introduce relevant methodologies in the biochemical sciences by uniquely integrating the theories and practices that drive the fields of biology, biotechnology and medicine. Methodologies have improved tremendously over the past ten years and new strategies and protocols that once were just applied by a few pioneering laboratories are now applied routinely, accompanied by ever more fluent boundaries between core disciplines – leading to what is now called the life sciences. In this eighth edition, all core methodologies covered in the previous edition have been kept and appropriately updated and consolidated. New chapters have been added to address the requirements of today’s students and scientists, who operate in a much broader area than did the typical biochemists one or two decades ago. The contents of this text are thus structured into six areas, all of which play pivotal roles in current research: basic principles, biochemistry and molecular biology, biophysical methods, information technology, -omics methods and chemical biology. Of course, due to space restraints that help to keep this text accessible, the addition of new material required us to consolidate and carefully select content to be kept from the previous edition. These decisions have been guided by the over-arching theme of this text, namely to present principles and techniques. Importantly, our experience as teachers has always been that many undergraduates are challenged by quantitative calcula- tions based on these principles, and hence we have continued the tradition of this text by including relevant mathematical and numerical tools as well as examples. Indeed, new chapters on data processing, visualisation and Python have example applications that can be accessed from the CUP website to aid understanding. Sadly, two authors of previous editions have passed away: John Fyffe died shortly after the seventh edition was launched and Alastair Aitken passed away in 2014. Also, Keith Wilson, John Walker and Robert Burns decided to retire, and we thus invited several new authors to contribute to this eighth edition. We are grateful to the authors and publishers who have granted us permission to reproduce and adapt their figures. We further wish to express our gratitude to Sonja Biberacher, Madeleine Dallaston, Emma Klepzig, Hannah Leeson and Megan Cross who have helped tremendously with preparing figures and photographs. The manuscript and figures for this book have been compiled entirely with open-source and academic software under Linux, and we would like to acknowledge the efforts of software developers and programmers who make their products freely available. xv xvi Preface Finally, our sincere thanks also extend to Katrina Halliday and her team at Cambridge University Press whose invaluable support has made it possible to produce this new edition. We welcome constructive comments from all students who use this book within their studies, and from teachers and academics who adopt the book to complement their courses. A ND R E A S H O F MA N N A N D S A MU E L C LO K IE CONTRIBUTORS PA R I SA A MA NI Structural Chemistry Program, Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland, Australia A N WA R R. BAY D OU N School of Life Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK DAV I D C A MP School of Environment, Griffith University, Nathan, Queensland, Australia C I N Z I A C A NTAC E SS I Cambridge Veterinary School, University of Cambridge, Cambridge, UK J E A N- BA PT I ST E C A Z I E R Centre for Computational Biology, University of Birmingham, Edgbaston, Birmingham, UK S A M U E L C LOK I E Bioinformatics Department, West Midlands Regional Genetics Laboratory Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK M E G AN C R OS S Structural Chemistry Program, Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland, Australia xvii xviii List of Contributors KAT J A F I SC H E R Scabies Laboratory, QIMR Berghofer, Locked Bag 2000, Royal Brisbane Hospital, Herston, Queensland, Australia RO B I N B. GA S S E R Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia J O H N GR A I NGE R Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK S T EP HE N E. H A R D I NG School of Biosciences, University of Nottingham, Sutton Bonington, UK SONJA HESS Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, USA A N D R E A S H OF MA NN Structural Chemistry Program, Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland, Australia and Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia J OA N NE KONK E L Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK PA S I K. KOR H ONE N Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia J OA N NE MAC D ONA LD School of Science and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland, Australia and  List of Contributors xix Department of Medicine, Columbia University, New York, NY, USA J A M E S I. MAC R A E Metabolomics Unit, The Francis Crick Institute, London, UK KAY O H LE ND I E C K Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland S T EP HE N W. PA D D OC K Howard Hughes Medical Institute, Department of Molecular Biology, University of Wisconsin, Madison, Wisconsin, USA A N N A V. P R OTA S I O Wellcome Trust Sanger Institute, University of Cambridge, Hinxton, Cambridge, UK RA L P H R A P LE Y Department of Biosciences, University of Hertfordshire, Hatfield, Hertfordshire, UK G I L L R U MSB Y Clinical Biochemistry, HSL Analytics LLP, London, UK A N N E S I MON Université Claude Bernard Lyon 1, Bâtiment Curien, Villeurbanne, France and Laboratoire Chimie et Biologie des Membranes et des Nanoobjets, Université de Bordeaux, Pessac, France RO B E RT J. S LAT E R Royal Society of Biology, Charles Darwin House, London, UK xx List of Contributors T I M J. S T E V E NS MRC Laboratory of Molecular Biology, University of Cambridge, Cambridge, UK PAUL TAY LOR School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, UK M I C H A E L W E I SS Phase I Pilot Consortium, Children’s Oncology Group, Monrovia, CA, USA 0 Tables and Resources Table 0.1 Abbreviations ADP adenosine 5′-diphosphate AMP adenosine 5′-monophosphate ATP adenosine 5′-triphosphate bp base pairs cAMP cyclic AMP CAPS N-cyclohexyl-3-aminopropanesulfonic acid CHAPS 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid cpm counts per minute CTP cytidine triphosphate DDT 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethane DMSO dimethylsulfoxide DNA deoxyribonucleic acid e− electron EDTA ethylenediaminetetra-acetate ELISA enzyme-linked immunosorbent assay FACS fluorescence-activated cell sorting FAD flavin adenine dinucleotide (oxidised) FADH2 flavin adenine dinucleotide (reduced) FMN flavin mononucleotide (oxidised) FMNH2 flavin mononucleotide (reduced) GC gas chromatography GTP guanosine triphosphate HAT hypoxanthine, aminopterin, thymidine medium HEPES 4(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid HPLC high-performance liquid chromatography IMS industrial methylated spirit kb kilobase pairs xxi xxii Tables and Resources Table 0.1 (cont.) Mr relative molecular mass MES 4-morpholine-ethanesulfonic acid min minute MOPS 4-morpholine-propanesulfonic acid NAD + nicotinamide adenine dinucleotide (oxidised) NADH nicotinamide adenine dinucleotide (reduced) NADP+ nicotinamide adenine dinucleotide phosphate (oxidised) NADPH nicotinamide adenine dinucleotide phosphate (reduced) PIPES 1,4-piperazinebis(ethanesulfonic acid) ppb parts per billion ppm parts per million RNA ribonucleic acid rpm revolutions per minute SDS sodium dodecyl sulfate TAPS N-[tris-(hydroxymethyl)]-3-aminopropanesulfonic acid TRIS 2-amino-2-hydroxymethylpropane-1,3-diol v/v volume per volume w/v weight per volume Table 0.2 Biochemical constants Unit Symbol SI equivalent Atomic mass unit u 1.661 × 10−27 kg Avogadro constant L or NA 6.022 × 1023 mol−1 Faraday constant F 9.648 × 104 C mol−1 Planck constant h 6.626 × 10−34 J s Universal or molar gas constant R 8.314 J K−1 mol−1 Molar volume of an ideal gas at standard conditionsa Vm 22.41 dm3 mol−1 Velocity of light in a vacuum c 2.997 × 108 m s−1 Note: aStandard conditions: pØ = 1 bar, θnormal = 25 °C.  Tables and Resources xxiii Table 0.3 SI units: basic and derived units Symbol (basic Definition of SI Equivalent Quantity SI unit SI units) unit in SI units Basic units Length metre m Mass kilogram kg Time second s Electric current ampere A Temperature kelvin K Luminous intensity candela cd Amount of substance mole mol Derived units Area square metre m2 Concentration mole per cubic metre mol m−3 Density kilogram per cubic kg m−3 metre Electric charge coulomb C 1As 1 J V−1 Electric potential volt V 1 kg m2 s−3A−1 1 J C−1 difference Electric resistance ohm Ω 1 kg m2 s−3A−2 1 V A−1 Energy, work, heat joule J 1 kg m2 s−2 1Nm Enzymatic activity katal katal 1 mol s −1 60 mol min−1 Force newton N 1 kg m s−2 1 J m−1 Frequency hertz Hz 1 s−1 Magnetic flux density tesla T 1 kg s−2 A−1 1 V s m−2 Molecular mass atomic mass units u 1 u = 1 Da (dalton) Molar mass gram per mole g mol−1 1 g mol−1 Power, radiant flux watt W 1 kg m2 s−3 1 J s−1 Pressure pascal Pa 1 kg m1 s−2 1 N m−2 Volume cubic metre m3 xxiv Tables and Resources Table 0.4 Conversion factors for non-SI units Unit Symbol SI equivalent Energy calorie cal 4.184 J erg erg 10−7 J electron volt eV 1.602 × 10−19 J Pressure atmosphere atm 1.013 × 105 Pa bar bar 105 Pa millimetres of mercury mm Hg (Torr) 133.322 Pa pounds per square inch psi 6.895×104 Pa Temperature degree Celsius ºC  θ + 273.15  K 1° C  degree Fahrenheit ºF  t  5   1° F − 32 × 9 + 273.15  K Length ångstrøm Å 10−10 m inch in 0.0254 m Mass pound lb 0.4536 kg Table 0.5 Common unit prefixes associated with quantitative terms Multiple Prefix Symbol Multiple Prefix Symbol 1024 yotta Y 10−1 deci d 1021 zetta Z 10−2 centi c 1018 exa E 10−3 milli m 1015 peta P 10−6 micro μ 1012 tera T 10−9 nano n 109 giga G 10−12 pico p 106 mega M 10−15 femto f 103 kilo k 10−18 atto a 10 2 hecto h 10 −21 zepto z 10 1 deca da 10 −24 yocto y  Tables and Resources xxv Table 0.6 Interconversion of non-SI and SI units of volume Non-SI unit Non-SI subunit SI subunit SI unit 1 litre (l) = 103 ml = 1 dm3 = 10−3 m3 1 millilitre (ml) = 1 ml = 1 cm 3 = 10−6 m3 1 microlitre (μl) = 10−3 ml = 1 mm3 = 10−9 m3 1 nanolitre (nl) = 10−6 ml = 1 nm3 = 10−12 m3 Table 0.7 Interconversion of mol, mmol and μmol in different volumes to give different concentrations Molar (M) Millimolar (mM) Micromolar (μM) 1 mol dm−3 = 103 mmol dm−3 = 106 μmol dm−3 = = = 1 mmol cm −3 = 10 μmol cm 3 −3 = 106 nmol cm−3 = = = 1 μmol mm −3 = 10 nmol mm 3 −3 = 106 pmol mm−3 Table 0.8 pKa values of some acids and bases that are commonly used as buffer solutions Acid or base pKa Acetic acid 4.8 Barbituric acid 4.0 Carbonic acid 6.1, 10.2 CAPSa 10.4 Citric acid 3.1, 4.8, 6.4 Glycylglycine 3.1, 8.1 HEPES a 7.5 MESa 6.1 MOPS a 7.1 Phosphoric acid 2.0, 7.1, 12.3 Phthalic acid 2.8, 5.5 PIPES a 6.8 Succinic acid 4.2, 5.6 TAPS a 8.4 Tartaric acid 3.0, 4.2 TRIS a 8.1 Note: aSee list of abbreviations (Table 0.1). xxvi Tables and Resources Table 0.9 Abbreviations for amino acids and average molecular masses, free and within peptides (residue mass) Molecular Residue mass mass M M-M(H2O) Three- One- letter letter Nominal Average Monoisotopic Amino acid code code mass (Da) mass (Da) mass (Da) Side Chain Alanine Ala A 89 71.08 71.037114 HO O H3 C NH2 HO O Arginine Arg R 174 156.19 156.10111 H 2N N NH2 NH HO O Asparagine Asn N 132 114.10 114.04293 O H2 N NH2 HO O Aspartic acid Asp D 133 115.09 115.02694 O HO NH2 Asparagine or Asx B aspartic acid HO O Cysteine Cys C 121 103.15 103.00919 HS NH2 HO O Glutamine Gln Q 146 128.13 128.05858 H 2N NH2 O HO O Glutamic acid Glu E 147 129.12 129.04259 HO NH2 O Glutamine or Glx Z glutamic acid HO O Glycine Gly G 75 57.05 57.021464 H NH2 HO O HN Histidine His H 155 137.14 137.05891 N NH2 HO O Isoleucine Ile I 131 113.16 113.08406 NH2 HO O Leucine Leu L 131 113.16 113.08406 NH2  Tables and Resources xxvii Table 0.9 (cont.) Molecular Residue mass mass M M-M(H2O) Three- One- letter letter Nominal Average Monoisotopic Amino acid code code mass (Da) mass (Da) mass (Da) Side Chain Lysine Lys K 146 128.17 128.09496 HO O H2N NH2 Methionine Met M 149 131.20 131.04048 HO O S NH2 Met-sulfoxide a MetSO MSO 165 147.20 147.03540 HO O S NH2 O Phenylalanine Phe F 165 147.18 147.06841 HO O NH2 Proline Pro P 115 97.12 97.052764 HO O NH Serine Ser S 105 87.08 87.032029 HO O HO NH2 Threonine Thr T 119 101.11 101.04768 HO O HO NH2 Tryptophan Trp W 204 186.21 186.07931 HO O HN NH2 HO HO O Tyrosine Tyr Y 181 163.18 163.06333 NH2 Valine Val V 117 99.13 99.068414 HO O NH2 Notes: Although some amino acids are similar in mass, they can be distinguished by modern high- resolution mass spectrometry. aThis is a frequently found modification in mass spectrometric investigation of proteins and peptides. Table 0.10 Ionisable groups found in amino acids pKa1 pKa2 pKa3 α-carboxyl group α-ammonium ion side-chain group R R R R OH O OH OH + H+ H2N H2N + H+ H3N H2N Amino acid O O O O pI Alanine 2.3 9.7 - 6.0 H2N NH2 H2N NH HN HN Arginine 2.2 9.0 12.5 10.8 OH OH H2N H2N + H+ O O Asparagine 2.0 8.8 - 5.41 OH O O Aspartic acid 1.9 9.6 3.7 O 2.8 OH OH H2N H2N + H+ O O HS S Cysteine 2.0 8.2 8.4 H2N OH OH + 5.1 H2N H+ O O Glutamine 2.2 9.1 - 5.7 HO O O O Glutamic acid 2.2 9.7 4.3 OH 3.2 OH H2N H2N + H+ O O Glycine 2.3 9.6 - 6.0 NH N HN HN Histidine 1.8 9.2 6.0 7.6 OH OH H2N H2O + H+ O O Isoleucine 2.4 9.6 - 6.0 Leucine 2.4 9.6 - 6.0 NH3 NH2 Lysine 2.2 9.0 10.5 9.7 OH OH H2N H2N + H+ O O Methionine 2.3 9.2 - 5.7 Phenylalanine 1.8 9.1 - 5.5 HO O HO O Proline 2.0 10.6 + H+ - 6.3 NH2 NH Serine 2.2 9.2 - 5.7 Threonine 2.1 9.1 - 5.6 Tryptophan 2.8 9.4 - 5.9 HO O Tyrosine 2.2 9.1 10.1 OH 5.7 OH H2N H2N + H+ O O Valine 2.3 9.6 - 6.0 xxx Tables and Resources Table 0.11 The genetic code: triplet codons and their corresponding amino acids U C A G UUU Phe UCU Ser UAU Tyr UGU Cys U UUC Phe UCC Ser UAC Tyr UGC Cys C U UUA Leu UCA Ser UAA Stop UGA Stop A UUG Leu UCG Ser UAG Stop UGG Trp G CUU Leu CCU Pro CAU His CGU Arg U CUC Leu CCC Pro CAC His CGC Arg C C CUA Leu CCA Pro CAA Gln CGA Arg A CUG Leu CCG Pro CAG Gln CGG Arg G AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C A AUA Ile ACA Thr AAA Lys AGA Arg A AUG Met ACG Thr AAG Lys AGG Arg G GUU Val GCU Ala GAU Asp GGU Gly U GUC Val GCC Ala GAC Asp GGC Gly C G GUA Val GCA Ala GAA Glu GGA Gly A GUG Val GCG Ala GAG Glu GGG Gly G  Biochemical and Molecular Biological 1 Methods in Life Sciences Studies SAMUEL CLOKIE AND ANDREAS HOFMANN 1.1 From Biochemistry and Molecular Biology to the Life Sciences 1 1.2 The Education of Life Scientists 2 1.3 Aims of Life Science Studies 3 1.4 Personal Qualities and Scientific Conduct 5 1.5 Suggestions for Further Reading 7 1.1 FROM BIOCHEMISTRY AND MOLECULAR BIOLOGY TO THE LIFE SCIENCES Biochemistry is a discipline in the natural sciences that is chiefly concerned with the chemical processes that take place in living organisms. Starting in the 1950s, a new stream evolved from traditional biochemistry, which, until then, mainly investigated bulk behaviour and macroscopic phenomena. This new stream focussed on the molec- ular basis of biological processes and since it put the biologically important molecules into the spotlight, the term molecular biology was coined. Importantly, molecular biology goes beyond the mere characterisation of molecules. It includes the study of interactions between biologically relevant molecules with the clear goal to reveal insights into functions and processes, such as replication, transcription and transla- tion of genetic material. Major technological and methodological advances made during the 1980s enabled the development and establishment of several specialised areas in the life sciences. These include structural biology (in particular the determination of three-dimensional structures), genetics (for example DNA sequencing) and proteomics. The refinement and improvement of methodologies, as well as the development of more efficient software (in line with more powerful computing resources), contributed substantially to specialist techniques becoming more accessible to researchers in neighbouring disciplines. What once had been the task of a highly specialised scientist who had been extensively trained in that particular area has consistently been transformed into a routinely applied methodology. These tendencies have pushed the feasibility of cross-disciplinary studies into an entirely new realm, and in many contemporary laboratories and research groups, methods originally at home in different basic disci- plines are frequently used next to each other. 1 2 Biochemical and Molecular Biological Methods in Life Sciences Studies It is thus not surprising, that in the past 15–20 years, the term ‘life sciences’ has been used to describe the general nature of studies and research areas of a scientist working on studies related to living organisms. 1.2 THE EDUCATION OF LIFE SCIENTISTS The life sciences embrace different fields of the natural and health sciences, all of which involve the study of living organisms, from microorganisms, plants and animals to human beings. Importantly, even satellite areas that are method- ologically rooted outside natural or health sciences, for example bioethics, have become a part of the life sciences. And with neuroscience and artificial intelligence being two major current areas of interest, one might expect further subjects to be included under the life sciences umbrella. The growing complexity of scientific studies requires ever-increasing cross-disciplinarity when it comes to particular methodologies utilised in the quest to reach the goals of these studies. This poses entirely new problems when it comes to the education of future scientists and researchers. At the core, this requires an appreciation or even acquisition of mindsets from other disciplines, as opposed to a mere concatenation of methods specific to one discipline. Therefore, the achievement of true cross-disciplinarity poses particular challenges since, according to Simon Penny, it requires deep professional humility, intellectual rigour and courage. At the same time, due to time and practical constraints, educational programmes that teach life science studies often focus on a select group of individual disciplines, rather than attempting to include every subject that could be included under the global term ‘life sciences’. In order to successfully embark on a contemporary life science study or contribute to large cross-disciplinary teams, scientists also need to possess knowledge and skills in a diverse range of areas. For example, in order to screen a small-molecule com- pound library against a target protein, the chemist needs to understand the nature and behaviour of proteins. Vice versa, if a cell biologist wants to screen a small-molecule library to identify novel effectors for a pathway of interest, they need to deal with the logistics and characteristics of small molecules. One relatively new aspect that the life scientist is faced with is the large amount of data generated by improved instrumentation and methodologies. The term ‘bioin- formatics’ has been coined that loosely describes the processing of biological data. It is now considered a stand-alone discipline that includes methods on processing ‘big data’ that are commonplace in the field of genomics. Such data-processing tech- niques are ubiquitous in the life sciences, and bioinformatics serves as an example that spans almost all the life science disciplines. Life scientists work in many diverse areas, including hospitals, academic teaching and research, drug discovery and development, agriculture, food institutes, general education, cosmetics and forensics. Aside from their specialist knowledge of the inter- facing of core disciplines, they also require a solid basis of transferable skills, such as analytical and problem-solving capabilities, and written and verbal communication, as well as planning, research, observation and numerical skills.  1.3 Aims of Life Science Studies 3 1.3 AIMS OF LIFE SCIENCE STUDIES Studies in the life sciences ultimately aspire to an advanced understanding of the nature of life in molecular and mechanistic terms. Biochemistry still constitutes the core discipline of the experimental life sciences, and involves the study of the chem- ical processes that occur in living organisms. Such studies rely on the application of appropriate techniques to advance our understanding of the nature, and relationships between, biological molecules, especially proteins and nucleic acids in the context of cellular function. The huge advances made in the past 10–15 years, with the Human Genome Project being a particular milestone, have stimulated major developments in our understand- ing of many diseases and led to identification of strategies that might be used to combat these diseases. Such progress was accompanied – and enabled – by substan- tial developments in technologies, data acquisition and data mining. For example, the genome of any living organism includes coding regions that are transcribed into messenger RNAs (mRNAs), which are subsequently translated into proteins. In vitro, mRNAs are reverse-transcribed (Section 4.10.4), resulting in stable complementary DNA, which is traditionally sequenced using a DNA polymerase, an oligonucleotide primer and four deoxyribonucleotide triphosphates (dNTPs) to synthesise the com- plementary strand to the template sequence (see Section 20.2.1). The development of high-throughput sequencing (‘next-generation sequencing’) technologies that can produce millions or billions of sequences concurrently, has made the sequencing of entire genomes orders of magnitude faster and, at the same time, less expensive. This particular development has made it possible to sequence many thousands of human genomes, making it possible to truly understand population genetics. Such informa- tion can be used to aid and improve genetic diagnosis of common and rare human diseases. The combination of molecular biology and genomics applied to the benefit of humankind can be best illustrated by the invention and recent improvement of genome editing techniques, such as the CRISPR/Cas method (see Section 4.17.6). The potential impact on health economics is substantial; individually unpleasant and costly conditions, genetically or environmentally acquired, can be addressed and potentially be eradicated. Similar developments have occurred in many other disciplines. In structural biology, robotics, especially at synchrotron facilities, have drastically reduced the time required for handling individual samples. Plate readers that perform particular spectroscopic applications (see Chapter 13) in a multi-well format are now ubiquitously present in laboratories and enable medium and high throughput for many standard assays (see also Section 24.5.3). All these developments are accompanied by a massive increase in (digital) data generated, which opens an avenue for entirely new types of studies that are more or less exclusively concerned with data mining and analysis. This text aims to cover the principles and methodologies underpinning life science studies and thus address the requirements of today’s students and scientists who operate in a much broader area than the typical biochemists one or two decades ago. 4 Biochemical and Molecular Biological Methods in Life Sciences Studies The contents of this text are therefore structured around six different disciplines or methodologies, all of which play pivotal roles in current research: Basic Principles (Chapter 2) Biochemistry and Molecular Biology (Chapters 3–10) Biophysics (Chapters 11–15) Information Technology (Chapters 16–19) ‘omics Methods (Chapters 20–22) Chemical Biology (Chapters 23–24) The Basic Principles chapter introduces some important general concepts surround- ing biologically relevant molecules, as well as their handling in aqueous solution. It further highlights fundamental considerations when designing and conducting exper- imental research. Information technology has become an integral part of scientific research. The abil- ity to handle, analyse and visualise data has always been a core skill of science and gained even more importance with the advent of ‘big data’ on the one hand, and the fact that data acquisition, processing and communication is done entirely in digital format on the other. Furthermore, in the areas of bio- and chemoinformatics, stan- dardisation of data formats has resulted in the availability of unprecedented volumes of information in databases that require an appropriate understanding in order to fully utilise the data resource. Among the core methodologies of biochemistry and molecular biology are tech- niques to culture living cells and microorganisms, and the preparation and handling of DNA, as well as the production and purification of proteins. Such experimental work is frequently accompanied by analytical or preparative gel electrophoresis, the use of antibodies (immunochemistry) or radio isotopes. In the medical sciences, a diagnostic test requires the application of biochemical or molecular techniques in a regulated environment, comprising the area of clinical biochemistry (discussed in Chapter 10). The characterisation of cells and molecules, as well as their interactions and pro- cesses, involves an array of biophysical techniques. Such techniques apply gravita- tional (centrifugation) or electrical forces (mass spectrometry), as well as interactions with light over a broad range of energy. The neologism ‘omics is frequently being used when referring to methodologies that characterise large pools of biologically relevant molecules, such as DNA (genome), mRNA (transcriptome), proteins (proteome) or metabolic products (metabolome). The first application of these methodologies were mainly concerned with the acquisition and collection of large datasets specific to one experiment or biological question. However, several areas of the life sciences now leave behind the phase of pure obser- vation and are increasingly applied to study the dynamics of an organism, a so-called systems biology approach. A hallmark of chemical biology is the use of small molecules, either purified or synthetically derived natural products or purpose-designed chemicals, to study the modulation of biological systems. The use of small-molecule compounds can be either  1.4 Personal Qualities and Scientific Conduct 5 exploratory in nature (probes) or geared towards therapeutic use where the desired activities of the compounds are to either activate or inhibit a target protein, which is typically, but not necessarily, an enzyme. Due to the specific role of a target protein in a given cellular pathway, the molecular interaction will ideally lead to modulation of processes involved in pathogenic situations. Even if a small molecule has a great number of side effects, it can be used as a probe and be useful to delineate molecular pathways in vitro. Given the breadth of topics, methodologies and applications, selections as to the individual contents presented in this text had to be made. The topics selected for this text have been carefully chosen to provide undergraduate students and non-specialist researchers with a solid overview of what we feel are the most relevant and funda- mental techniques. Methods and techniques form the tool set of an experimental scientist. They are applied in the context of studies which, in the life sciences, address questions of the following nature: the structure and function of the total protein component of the cell (proteomics) and of all the small molecules in the cell (metabolomics) the mechanisms involved in the control of gene such expression the identification of genes associated with a wide range of diseases and the develop- ment of gene therapy strategies for the treatment of diseases the characterisation of the large number of ‘orphan’ receptors, whose physiological role and natural agonist are currently unknown, present in the host and pathogen genomes and their exploitation for the development of new therapeutic agents the identification of novel disease-specific markers for the improvement of clinical diagnosis the engineering of cells, especially stem cells, to treat human diseases the understanding of the functioning of the immune system in order to develop strategies for protection against invading pathogens the development of our knowledge of the molecular biology of plants in order to engineer crop improvements, pathogen resistance and stress tolerance the discovery of novel therapeutics (drugs and vaccines) to the nature and treatment of bacterial, fungal and viral diseases. 1.4 PERSONAL QUALITIES AND SCIENTIFIC CONDUCT The type of tasks in scientific research and the often long-term goals pursued by science require, very much like any other profession, particular attributes of people working in this area: Quite obviously, a substantial level of intelligence is required to grasp the scientific concepts in the area of study. The ability to think with clarity and logically, and to transform particular observations (low level of abstraction) into concepts (high level of abstraction) is also required, as is a solid knowledge of the basic mathematical syllabus for any area of the life sciences. 6 Biochemical and Molecular Biological Methods in Life Sciences Studies For pursuing longer-term goals it is also necessary to possess stamina and persistence. Hurdles and problems need to be overcome, and failures need to be coped with. Often, experimental series can become repetitive and it is important to not fall into the trap of boredom (and then become negligent). A frequently underestimated quality is attention to detail. Science and research is about getting things right. At the time when findings from research projects are writ- ten up for publication, or knowledge is summarised for text books such as this one, the readership and the public expect that all details are correct. Of course, mistakes can and do happen, but they should not happen commonly, and practices need to be in place that prevent mistakes being carried over to the next step. Critical self-ap- praisal and constant attention to detail is probably the most important element in this process. Communication skills and the ability to describe and visualise fairly specialist con- cepts to non-experts is of great importance as well. The best set of data is not put to good use if it is not presented to the right forum in the right fashion. Likewise, any set of knowledge acquired cannot be taught effectively to others without such skills. Lastly, curiosity and the willingness to explore are a requirement if an independent career in the life sciences is being sought. Just having excellent marks in science sub- jects does not automatically make a scientist if one needs to be told every single next step through a research project. Since science does not happen in an isolated situation but a community (the scien- tific community on the one hand, but also society as a whole on the other), norms need to be put in place that define and guide what is acceptable and unacceptable behaviour. Beset by the occasional fraudulent study and scientific misconduct case, more attention has been paid to ethical conduct in science in the recent decade (see also Section 2.8.2). Despite ethical frameworks and policies put in place to varying degrees, ultimately, the responsibility of ethical conduct rests with the individual. And while many of the ethical norms are geared towards the interactions of scientists when it comes to specific scientific tasks or procedures, there are certainly elements that apply to any (scientific or non-scientific) situation: Critically assess potential conflicts of interest. In a surprisingly large number of situa- tions, any individual might find themselves playing multiple roles, and the objectives of each of the roles may be in conflict with each other. Where conflicts of interest arise, the individual should withdraw from the decision-making process. Respect confidentiality and privacy. This not only applies to ongoing research work, results, etc., but also to conversations and advice. When approached for advice or with a personal conversation, most parties expect this to be treated in confidence and not made available to the public. Follow informed consent rules and discuss intellectual property frankly. In order to avoid disagreements about who should get credit and for which aspects, talk about these issues at the beginning of a working relationship. Engage in a sharing culture. Resources, methodologies, knowledge and skills that have been established should be shared where reasonably possible. This fosters positive  1.5 Suggestions for Further Reading 7 interactions, contributes to transparency and frequently leads to new collaborations, all of which advance science. Lead by example. Not just in a teaching situation, but in most other settings, too, gen- eral principles and cultural norms are only effectively imprinted into the environment if the rules and guidelines are lived. In many instances, rules and protocols are ‘imple- mented’ by institutions and exist mainly as a ticking-the-box exercise. Such policies do not address the real point as they should and are largely ineffective. 1.5 SUGGESTIONS FOR FURTHER READING 1.5.1 Experimental Protocols Holmes D., Moody P. and Dine D. (2010) Research Methods for the Biosciences, 2nd Edn., Oxford University Press, Oxford, UK. 1.5.2 General Texts Smith D. (2003) Five principles for research ethics. Monitor on Psychology 34, 56. 1.5.3 Review Articles Duke C.S. and Porter J.H. (2013) The ethics of data sharing and reuse in biology. BioScience 63, 483–489. Puniewska M. (2014) Scientists have a sharing problem. The Atlantic, 15 Dec 2014, www.theatlantic.com/health/archive/2014/12/scientists-have-a-sharing- problem/383061/ (accessed April 2017). Taylor P.L. (2007) Research sharing, ethics and public benefit. Nature Biotechnology 25, 398–401. 1.5.4 Websites Mike Brotherton’s Blog: Five qualities required to be a scientist www.mikebrotherton.com/2007/11/05/five-qualities-required-to-be-a-scientist/ (accessed April 2017) Simon Penny: Rigorous Interdisciplinary Pedagogy simonpenny.net/texts/rip.html (accessed April 2017) Andy Polaine’s Blog: Interdisciplinarity vs Cross-Disciplinarity www.polaine.com/2010/06/interdisciplinarity-vs-cross-disciplinarity/ (accessed April 2017)  2 Basic Principles PARISA AMANI AND ANDREAS HOFMANN 2.1 Biologically Important Molecules 8 2.2 The Importance of Structure 9 2.3 Parameters of Biological Samples 18 2.4 Measurement of the pH: The pH Electrode 24 2.5 Buffers 26 2.6 Ionisation Properties of Amino Acids 30 2.7 Quantitative Biochemical Measurements 32 2.8 Experiment Design and Research Conduct 33 2.9 Suggestions for Further Reading 38 2.1 BIOLOGICALLY IMPORTANT MOLECULES Molecules of biological interest can be classified into ions, small molecules and macromolecules. Typical organic small molecules include the ligands of enzymes, substrates such as adenosine triphosphate (ATP) and effector molecules (inhibitors, drugs). Ions such as Ca2+ play a key role in signalling events. Biological macromolecules are polymers which, by definition, consist of covalently linked monomers, the building blocks. The four types of biologically relevant polymers are summarised in Table 2.1. 2.1.1 Proteins Proteins are formed by a condensation reaction of the α-amino group of one amino acid (or the imino group of proline) with the α-carboxyl group of another. Concomitantly, a water molecule is lost and a peptide bond is formed. The pep- tide bond possesses partial double-bond character and thus restricts rotation around the C–N bond. The progressive condensation of many amino acids gives rise to an unbranched polypeptide chain. Since biosynthesis of proteins proceeds from the N- to the C-terminal amino acid, the N-terminal amino acid is taken as the beginning of the chain and the C-terminal amino acid as the end. Generally, chains of amino acids containing fewer than 50 residues are referred to as peptides, and those with more than 50 are referred to as proteins. Most proteins contain many hundreds of amino acids; ribonuclease, for example, is considered an extremely small protein with only 103 amino-acid residues. Many biologically active peptides contain 20 or fewer amino 8  2.2 The Importance of Structure 9 Table 2.1 The four types of biologically relevant polymers Polymer Monomers Monomer details Ribonucleic acid 4 Bases Adenine (A), uracil (U), cytosine (C), guanine (G) (RNA) Deoxyribonucleic 4 Bases Adenine (A), thymine (T), cytosine (C), acid (DNA) guanine (G) Protein 20 Amino acids Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr Polysaccharide Monosaccharides Trioses, tetroses, pentoses, hexoses, heptoses acids, such as the mammalian hormone oxytocin (nine amino-acid residues) which is clinically used to induce labour since it causes contraction of the uterus, and the neurotoxin apamin (18 amino-acid residues) found in bee venom. 2.2 THE IMPORTANCE OF STRUCTURE Three main factors determine the three-dimensional structure of a macromolecule: allowable backbone angles interactions between the monomeric building blocks interactions between solvent and macromolecule. The solvent interactions can be categorised into two types: binding of solvent mol- ecules (solvation) and hydrophobic interactions. The latter arise from the inabil- ity or reluctance of parts of the macromolecule to interact with solvent molecules (hydrophobic effect), which, as a consequence, leads to exclusive solvent–solvent interactions. Phenomenologically, a collection of molecules that cannot be solvated will stick close to one another and minimise solvent contact. The interactions between the building blocks of the macromolecule comprise nega- tive interactions (by avoiding atomic clashes) and positive interactions, which may be provided by hydrogen bonds, electrostatic interactions and van der Waals interactions (see also Section 17.4). Hydrogen bonds are the main constitutive force of back- bone interactions in proteins, but can also be observed between residue side chains. Electrostatic interactions in proteins occur between residue side chains that pos- sess opposite charges (arginine, lysine and aspartate, glutamate). The van der Waals attraction is a weak short-range force that occurs between all molecules. It becomes particularly important if two molecules possess highly complementary shapes. Thus, van der Waals interactions are responsible for producing complementary surfaces in appropriate regions of macromolecules. The allowable backbone angles provide a framework of geometric constraints and balance attractive interactions and geometrical/steric tension within the macromolecule. 10 Basic Principles 2.2.1 Conformation The structural arrangement of groups of atoms is called conformation (see also Section 17.1.1) and the conformational isomerism of molecules describes isomers that can be inter converted exclusively by rotations about formally single bonds. The rotation about a single bond is restricted by a rotational energy barrier that must be overcome; the individual isomers are called rotamers. Conformational isomerism arises when the energy barrier is small enough for the interconversion to occur. The angle describing the rotation around a bond between two atoms is called the dihe- dral (or torsion) angle. The protein backbone (Figure 2.1) is geometrically defined by three dihedral angles, namely Φ (N-Cα), Ψ (Cα-C) and ω (C-N); the last angle can take only two values, 0° and 180°, due to the partial double-bond character of the peptide bond. The conformation of the protein backbone is therefore determined by the Φ and Ψ torsion angles. The values these angles assume determine which type of secondary structure (see below) a certain consecutive region in the protein will adopt. Many organic small molecules possess cyclic aromatic structures and are thus pla- nar. However, there are also many non-aromatic cyclic structures, in particular car- bohydrates (sugars), which are of great importance in biochemical processes. Notably, many sugars exist in aqueous solution as both open-chain and cyclic forms. Figure 2.2 illustrates this using the example of D-glucose. It is obvious from the open-chain form, that there are a number of stereogenic centres in D-glucose. If the positions of the hydroxyl groups are changed to the opposite enantiomer for each stereogenic carbon, the resulting molecule is L-glucose. Upon ring closure of the open-chain form, atoms or groups bonded to tetrahedral ring carbons are either pointing up or down, as indicated by the use of dashed or solid wedges when drawing the two- dimensional structures. If two neighbouring hetero-atom substituents (e.g. hydroxyl groups) on the ring are both pointing in the same direction, this conformation is called cis; if they point in opposite directions, they are said to be in the trans confor- mation. The open-chain form is characterised by an aldehyde function which, upon ring closure, is converted to a hemi-acetal (comprising R1C(OR2)(OH)H; see the carbon Residue i + 1 Figure 2.1 Polypeptide chain comprising three amino acids (numbered i−1, i, i+1). The limits of a single residue Cα (i) are indicated by the dashed lines. The torsion angles Φ, O Ψ and ω describe the bond rotations around the N-Cα, ω Cα-C and C-N bonds, respectively. Residue i N ψ C Cβ H φ Cα H N C O Cα Residue i – 1  2.2 The Importance of Structure 11 surrounded by the blue and red highlighted oxygen atoms in Figure 2.2). Formation of the hemi-acetal can result in either α-D-glucose or β-D-glucose, depending on the position of the hydroxyl group in the anomeric position (highlighted in blue). In five- and six-membered non-aromatic ring structures, the bond angles are close to tetrahedral (109.5°) giving rise to a ring shape that is not flat. The lowest energy (and thus most stable) ring conformation is the so-called chair conformation (Figure 2.3). An alternate chair conformation is obtained through a process called ring inver- sion, if one of the ‘up’ carbon atoms moves downwards and one of the ‘down’ carbon atoms upwards. Another ring conformation of six-membered rings is the so-called boat conformation, where the substituents at the ‘bow’ and the ‘stern’ are close enough to each other to cause van der Waals repulsion. Therefore, the boat conforma- tion possesses higher energy and is hence less favoured than the chair conformation. 2.2.2 Folding and Structural Hierarchy Macromolecular molecules, due to their physical extents, have the ability to bend and therefore bring various regions of their atomic arrangements into contact with each other. This process and the resulting three-dimensional structure will be driven HO OH OH OH OH O OH O OH O HO HO OH HO OH HO H OH OH α-form β-form open-chain cyclic cis trans Figure 2.2 The open-chain and the cyclic forms of D-glucose shown as two-dimensional drawings as well as with their three-dimensional atomic structures. Ring closure of t