PHYSICAL BIOCHEMISTRY: PRINCIPLES AND APPLICATIONS

Transcription

1 PHYSICAL BIOCHEMISTRY: PRINCIPLES AND APPLICATIONS Second Edition David Sheehan Department of Biochemistry University College Cork Ireland A John Wiley & Sons, Ltd, Publication

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3 PHYSICAL BIOCHEMISTRY

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5 PHYSICAL BIOCHEMISTRY: PRINCIPLES AND APPLICATIONS Second Edition David Sheehan Department of Biochemistry University College Cork Ireland A John Wiley & Sons, Ltd, Publication

6 This edition first published 2009, C 2009 John Wiley & Sons Ltd Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley s global Scientific, Technical and Medical business with Blackwell Publishing. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Other Editorial Offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK 111 River Street, Hoboken, NJ , USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Sheehan, David, 1958 Physical biochemistry : principles and applications / David Sheehan. 2nd ed. p. ; cm. Includes bibliographical references and index. ISBN (hb) ISBN (pb) 1. Physical biochemistry. I. Title. [DNLM: 1. Biophysics. 2. Biochemistry. 3. Chemistry, Phisical. QT 34 S541p 2008] QD476.2.S dc ISBNs: (HB) (PB) A catalogue record for this book is available from the British Library. Set in 9/11pt Times by Aptara Inc., New Delhi, India Printed in Singapore by Fabulous Printing Pte Ltd First Impression 2009

7 To the memory of my father, Patrick Sheehan ( )

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9 Contents Preface xv Chapter 1 Introduction Special Chemical Requirements of Biomolecules Factors Affecting Analyte Structure and Stability ph Effects Temperature Effects Effects of Solvent Polarity Buffering Systems Used in Biochemistry How Does a Buffer Work? Some Common Buffers Additional Components Often Used in Buffers Quantitation, Units and Data Handling Units Used in the Text Quantification of Protein and Biological Activity The Worldwide Web as a Resource in Physical Biochemistry The Worldwide Web Web-Based Resources for Physical Biochemistry Objectives of this Volume 9 References 10 Chapter 2 Chromatography Principles of Chromatography The Partition Coefficient Phase Systems Used in Biochemistry Liquid Chromatography Gas Chromatography Performance Parameters Used in Chromatography Retention Resolution Physical Basis of Peak Broadening Plate Height Equation Capacity Factor Peak Symmetry Significance of Performance Criteria in Chromatography Chromatography Equipment Outline of Standard System Used Components of Chromatography System Stationary Phases Used Elution Modes of Chromatography Ion Exchange Gel Filtration Reversed Phase Hydrophobic Interaction Affinity 31

10 viii CONTENTS Immobilized Metal Affinity Chromatography Hydroxyapatite Open Column Chromatography Equipment Used Industrial Scale Chromatography of Proteins High Performance Liquid Chromatography (HPLC) Equipment Used Stationary Phases in HPLC Liquid Phases in HPLC Two Dimensional HPLC Fast Protein Liquid Chromatography Equipment Used Comparison with HPLC Perfusion Chromatography Theory of Perfusion Chromatography Practice of Perfusion Chromatography Membrane-Based Chromatography Systems Theoretical Basis Applications of Membrane-Based Separations Chromatography of a Sample Protein Designing a Purification Protocol Ion Exchange Chromatography of a Sample Protein: Glutathione Transferases HPLC of Peptides From Glutathione Transferases 50 References 50 Chapter 3 Spectroscopic Techniques The Nature of Light A Brief History of the Theories of Light Wave-Particle Duality Theory of Light The Electromagnetic Spectrum The Electromagnetic Spectrum Transitions in Spectroscopy Ultraviolet/Visible Absorption Spectroscopy Physical Basis Equipment Used in Absorption Spectroscopy Applications of Absorption Spectroscopy Fluorescence Spectroscopy Physical Basis of Fluorescence and Related Phenomena Measurement of Fluorescence and Chemiluminescence External Quenching of Fluorescence Uses of Fluorescence in Binding Studies Protein Folding Studies Resonance Energy Transfer Applications of Fluorescence in Cell Biology Spectroscopic Techniques Using Plane-Polarized Light Polarized Light Chirality in Biomolecules Circular Dichroism (CD) Equipment Used in CD CD of Biopolymers 81

11 CONTENTS ix Linear Dichroism (LD) LD of Biomolecules Infrared Spectroscopy Physical Basis of Infrared Spectroscopy Equipment Used in Infrared Spectroscopy Uses of Infrared Spectroscopy in Structure Determination Fourier Transform Infrared Spectroscopy Raman Infrared Spectroscopy Nuclear Magnetic Resonance (NMR) Spectroscopy Physical Basis of NMR Spectroscopy Effect of Atomic Identity on NMR The Chemical Shift Spin Coupling in NMR Measurement of NMR Spectra Electron Spin Resonance (ESR) Spectroscopy Physical Basis of ESR Spectroscopy Measurement of ESR Spectra Uses of ESR Spectroscopy in Biochemistry Lasers Origin of Laser Beams Some Uses of Laser Beams Surface Plasmon Resonance Equipment Used in SPR Use of SPR in Measurement of Adsorption Kinetics 107 References 110 Chapter 4 Mass Spectrometry Principles of Mass Spectrometry Physical Basis Overview of MS Experiment Ionization Modes Equipment Used in MS Analysis Mass Spectrometry of Proteins/Peptides Sample Preparation MS Modes Used in the Study of Proteins/Peptides Fragmentation of Proteins/Peptides in MS Systems Interfacing MS With other Methods MS/MS LC/MS GC/MS Electrophoresis/MS Uses of Mass Spectrometry in Biochemistry MS and Microheterogeneity in Proteins Confirmation and Analysis of Peptide Synthesis Peptide Mapping Post-Translational Modification Analysis of Proteins Determination of Protein Disulfide Patterns Protein Sequencing by MS Studies on Enzymes Analysis of DNA Components 139 References 143

12 x CONTENTS Chapter 5 Electrophoresis Principles of Electrophoresis Physical Basis Historical Development of Electrophoresis Gel Electrophoresis Nondenaturing Electrophoresis Polyacrylamide Nondenaturing Electrophoresis Protein Mass Determination by Nondenaturing Electrophoresis Activity Staining Zymograms Denaturing Electrophoresis SDS Polyacrylamide Gel Electrophoresis SDS Polyacrylamide Gel Electrophoresis in Reducing Conditions Chemical Crosslinking of Proteins Quaternary Structure Urea Electrophoresis Electrophoresis in DNA Sequencing Sanger Dideoxynucleotide Sequencing of DNA Sequencing of DNA Footprinting of DNA Single Strand Conformation Polymorphism Analysis of DNA Isoelectric Focusing (IEF) Ampholyte Structure Isoelectric Focusing Titration Curve Analysis Chromatofocusing Immunoelectrophoresis Dot Blotting and Immunodiffusion Tests with Antibodies Zone Electrophoresis/Immunodiffusion Immunoelectrophoresis Rocket Immunoelectrophoresis Counter Immunoelectrophoresis Crossed Immunoelectrophoresis (CIE) Agarose Gel Electrophoresis of Nucleic Acids Formation of an Agarose Gel Equipment for Agarose Gel Electrophoresis Agarose Gel Electrophoresis of DNA and RNA Detection of DNA and RNA in Gels Pulsed Field Gel Electrophoresis Physical Basis of Pulsed Field Gel Electrophoresis Equipment Used for Pulsed Field Gel Electrophoresis Applications of Pulsed Field Gel Electrophoresis Capillary Electrophoresis Physical Basis of Capillary Electrophoresis Equipment Used in Capillary Electrophoresis Variety of Formats in Capillary Electrophoresis Electroblotting Procedures Equipment Used in Electroblotting Western Blotting Southern Blotting of DNA Northern Blotting of RNA Blotting as a Preparative Procedure for Polypeptides 195

13 CONTENTS xi 5.11 Electroporation Transformation of Cells Physical Basis of Electroporation 196 References 196 Chapter 6 Three-Dimensional Structure Determination of Macromolecules The Protein-Folding Problem Proteins are only Marginally Stable Protein Folding as a Two-State Process Protein-Folding Pathways Chaperones Structure Determination by NMR Relaxation in One-Dimensional NMR The Nuclear Overhauser Effect (NOE) Correlation Spectroscopy (COSY) Nuclear Overhauser Effect Spectroscopy (NOESY) Sequential Assignment and Structure Elucidation Multi-Dimensional NMR Other Applications of Multi-Dimensional NMR Limitations and Advantages of Multi-Dimensional NMR Crystallization of Biomacromolecules What are Crystals? Symmetry in Crystals Physical Basis of Crystallization Crystallization Methods Mounting Crystals for Diffraction X-Ray Diffraction by Crystals X-Rays Diffraction of X-Rays by Crystals Bragg s Law Reciprocal Space Calculation of Electron Density Maps Calculation of Structure Factors Information Available from the Overall Diffraction Pattern The Phase Problem Isomorphous Replacement Molecular Replacement Anomalous Scattering Calculation of Electron Density Map Refinement of Structure Synchrotron Sources Other Diffraction Methods Neutron Diffraction Electron Diffraction Comparison of X-Ray Crystallography with Multi-Dimensional NMR Crystallography and NMR are Complementary Techniques Different Attributes of Crystallography- and NMR-derived Structures Structural Databases The Protein Database Finding a Protein Structure in the Database 257 References 259

14 xii CONTENTS Chapter 7 Hydrodynamic Methods Viscosity Definition of Viscosity Measurement of Viscosity Specific and Intrinsic Viscosity Dependence of Viscosity on Characteristics of Solute Sedimentation Physical Basis of Centrifugation The Svedberg Equation Equipment Used in Centrifugation Subcellular Fractionation Density Gradient Centrifugation Analytical Ultracentrifugation Sedimentation Velocity Analysis Sedimentation Equilibrium Analysis Methods for Varying Buffer Conditions Ultrafiltration Dialysis Precipitation Flow Cytometry Flow Cytometer Design Cell Sorting Detection Strategies in Flow Cytometry Parameters Measurable by Flow Cytometry 288 References and Further Reading 290 Chapter 8 Biocalorimetry The Main Thermodynamic Parameters Activation Energy of Reactions Enthalpy Entropy Free Energy Isothermal Titration Calorimetry Design of an Isothermal Titration Calorimetry Experiment ITC in Binding Experiments Changes in Heat Capacity Determined by Isothermal Titration Calorimetry Differential Scanning Calorimetry Outline Design of a Differential Scanning Calorimetry Experiment Applications of Differential Scanning Calorimetry Determination of Thermodynamic Parameters by Non-Calorimetric Means Equilibrium Constants 301 References 302 Chapter 9 Bioinformatics Overview of Bioinformatics Sequence Databases Nucleotide Sequence Databases 309

15 CONTENTS xiii Protein Sequence Databases Genome Databases Expressed Sequence Tag Databases Single Nucleotide Polymorphism (SNP) Database Tools for Analysis of Primary Structures BLAST Programs FastA Clustal W Hydropathy Plots Predicting Secondary Structure Identifying Protein Families Tertiary Structure Databases Cambridge Database Protein Databank (PDB) Specialist Structural Databases Programs for Analysis and Visualization of Tertiary Structure Databases Ras Mol/Ras Top Protein Explorer Py MOL Web Mol Swiss-Pdb Viewer Homology Modelling Modelling Proteins from Known Homologous Structures Automated Modelling Applications of Homology Modelling to Drug Discovery 346 References 346 Chapter 10 Proteomics Electrophoresis in Proteomics Two-Dimensional SDS PAGE Basis of 2-D SDS PAGE Equipment Used in 2-D SDS PAGE Analysis of Cell Proteins Free Flow Electrophoresis Blue Native Gel Electrophoresis Other Electrophoresis Methods Used in Proteomics Mass Spectrometry in Proteomics Tagging Methodologies Used in MS Proteomics Isotope-Coded Affinity Tagging (ICAT) for Cysteine-Containing Proteins Tagging of N- and C-Termini Tagging for Tandem MS Chip Technologies in Proteomics Microarrays Protein Biochips SELDI-TOF MS on Protein Chips Post-Translational Modification Proteomics Proteolysis Glycosylation Oxidation Protein Disulfides 374