Modern Biophysical Chemistry: Detection and Analysis of Biomolecules

This updated and up–to–date version of the first edition continues with the really interesting stuff to spice up a standard biophysics and biophysical chemistry course. All relevant methods used in current cutting edge research including such recent developments as super–resolution microscopy and next–generation DNA sequencing techniques, as well as industrial applications, are explained. The text has been developed from a graduate course taught by the author for several years, and by presenting a mix of basic theory and real–life examples, he closes the gap between theory and experiment. The first part, on basic biophysical chemistry, surveys fundamental and spectroscopic techniques as well as biomolecular properties that represent the modern standard and are also the basis for the more sophisticated technologies discussed later in the book. The second part covers the latest bioanalytical techniques such as the mentioned super–resolution and next generation sequencing methods, confocal fluorescence microscopy, light sheet microscopy, two–photon microscopy and ultrafast spectroscopy, single molecule optical, electrical and force measurements, fluorescence correlation spectroscopy, optical tweezers, quantum dots and DNA origami techniques. Both the text and illustrations have been prepared in a clear and accessible style, with extended and updated exercises (and their solutions) accompanying each chapter. Readers with a basic understanding of biochemistry and/or biophysics will quickly gain an overview of cutting edge technology for the biophysical analysis of proteins, nucleic acids and other biomolecules and their interactions. Equally, any student contemplating a career in the chemical, pharmaceutical or bio–industry will greatly benefit from the technological knowledge presented. Questions of differing complexity testing the reader’s understanding can be found at the end of each chapter with clearly described solutions available on the Wiley–VCH textbook homepage under: www.wiley–vch.de/textbooks Review of the first edition: “A fine introduction for physically oriented bioscientists at the graduate level or beyond” Chemistry World

Foreword to the Second Edition XIII Introduction 1 What is Biophysical Chemistry? – An Example from Drug Screening 1 Part One Basic Methods in Biophysical Chemistry 11 1 Basic Optical Principles 13 1.1 Introduction 13 1.2 What Does the Electronic Structure of Molecules Look Like? Orbitals, Wave Functions and Bonding Interactions 15 1.3 How Does Light Interact with Molecules? Transition Densities and the Transition Dipole Moment 20 1.4 Absorption Spectra of Molecules in Liquid Environments. Vibrational Excitation and the Franck–Condon Principle 24 1.5 What Happens After Molecules have Absorbed Light? Fluorescence, Nonradiative Transitions and the Triplet State 27 1.6 Quantitative Description of all Processes: Quantum Efficiencies, Kinetics of Excited State Populations and the Jablonski Diagram 33 Problems 38 Bibliography 39 2 Optical Properties of Biomolecules 41 2.1 Introduction 41 2.2 Experimental Determination of Absorption and Fluorescence Spectra 41 2.3 Optical Properties of Proteins and DNA 45 2.3.1 Intrinsic Absorption and Fluorescence of Amino Acids, Peptides and Proteins 45 2.3.2 Intrinsic Absorption of Nucleotides, DNA and RNA 47 2.4 Optical Properties of Important Cofactors 49 2.4.1 Haem 49 2.4.2 Nicotinamide Adenine Dinucleotides 52 2.4.3 Flavins 53 2.4.4 Chlorophylls 54 2.4.5 Carotenoids 56 Problems 58 Bibliography 58 3 Basic Fluorescence Techniques 61 3.1 Introduction 61 3.2 Fluorescent Labelling and Linking Techniques 61 3.2.1 Primary Amino Group Reactive Labels 63 3.2.2 Thiol Group Reactive Labels 64 3.2.3 Avidin–Biotin Techniques 65 3.2.4 His–Tag 66 3.2.5 Thiolinkers and Gold Surfaces 67 3.2.6 Fluorescent Proteins 67 3.3 Fluorescence Detection Techniques 68 3.4 Fluorescence Polarization Anisotropy 70 3.4.1 Principles and Theoretical Background 70 3.4.2 Application Example: Receptor–Ligand Interactions 78 3.4.3 Application Example: Estimation of Molecular Mass 79 3.4.4 Application Example: Enzyme Function and Kinetics 80 3.4.5 Application Example: Enzyme Inhibition, Activation and Regulation 83 3.5 Förster Resonance Energy Transfer 84 3.5.1 Principles and Theoretical Background 84 3.5.2 Application Examples 90 3.6 Fluorescence Kinetics 93 3.7 Fluorescence Recovery after Photobleaching 98 3.8 Biochemiluminescence 99 Problems 100 Bibliography 103 4 Chiroptical and Scattering Methods 105 4.1 Chiroptical Methods 105 4.1.1 Circular Dichroism (CD) 105 4.1.2 Optical Rotatory Dispersion 107 4.2 Light Scattering 109 4.2.1 Scattering of Light at Molecules Smaller than the Optical Wavelength 110 4.2.2 Scattering of Light at Particles Equal to or Larger than the Optical Wavelength 112 4.2.3 Dynamic Light Scattering 115 4.3 Vibrational Spectra of Biomolecules 115 Problems 118 Bibliography 119 5 Magnetic Resonance Techniques 121 5.1 Nuclear Magnetic Resonance of Biomolecules 121 5.1.1 Principles 121 5.1.2 Theoretical Framework 123 5.1.3 Primary Information Deduced from NMR Spectra 125 5.1.4 Pulsed NMR Spectroscopy 126 5.1.5 Two–Dimensional NMR Spectroscopy 130 5.1.6 Correlated Spectroscopy (COSY) 130 5.1.7 Nuclear Overhauser Effect and NOESY Spectra 135 5.1.8 NMR–Based Structural Analysis of Biomolecules 138 5.2 Electron Paramagnetic Resonance 141 Problems 145 Bibliography 147 6 Mass Spectrometry 149 6.1 Introduction 149 6.2 MALDI–TOF 149 6.2.1 Ionization 149 6.2.2 Analyser 152 6.2.3 Detector 153 6.2.4 Signals and Signal Improvements 154 6.3 ESI–MS 156 6.3.1 Ionization 156 6.3.2 Analyser and Detection 158 6.3.3 Signals and Signal Improvements 161 6.4 Structural and Sequence Analysis Using Mass Spectrometry 163 Problems 164 Bibliography 165 Part Two Advanced Methods in Biophysical Chemistry 167 7 Fluorescence Microscopy 169 7.1 Introduction 169 7.2 Conventional Fluorescence Microscopy 169 7.2.1 Confocal Fluorescence Microscopy 169 7.2.2 Laser Scanning Microscopy 174 7.2.3 Wide–Field Fluorescence Microscopy 174 7.3 Total Internal Reflection Fluorescence Microscopy 176 7.4 Light–Sheet Microscopy 178 Problems 180 Bibliography 181 8 Super–Resolution Fluorescence Microscopy 183 8.1 Stimulated Emission Depletion (STED) Microscopy 184 8.2 Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) 187 8.3 3D Super–Resolution Fluorescence Microscopy 190 8.3.1 3D–STED 190 8.3.2 3D–PALM/STORM 191 8.4 Imaging of Live Cells 191 8.4.1 Observation Duration 192 8.4.2 Irradiation Intensity 193 8.4.3 Imaging Depths 194 8.4.4 Labelling Conditions 194 8.5 Multicolour Super–Resolution Fluorescence Microscopy 195 8.6 Structured Illumination Microscopy 195 8.7 SOFI 197 8.8 Final Comparison 199 Problems 201 Bibliography 202 9 Single–Biomolecule Techniques 203 9.1 Introduction 203 9.2 Optical Single–Molecule Detection 203 9.2.1 Application Example 1: Observation of the Rotation of Single ATPase Complexes 206 9.2.2 Application Example 2: Single–Molecule Observation of the Elementary Steps of Biomolecular Motors 209 9.3 Fluorescence Correlation Spectroscopy 213 9.3.1 Autocorrelation Analysis and Observable Key Parameters 214 9.3.2 Autocorrelation Analysis, Mathematical Background 220 9.3.3 Quantitative Determination of Important Parameters from Autocorrelation Curves 221 9.3.3.1 Relationship Between Volume and Molecular Mass of a Protein and its Diffusion Time 222 9.3.3.2 Two–Dimensional Diffusion and Active Transport 222 9.3.3.3 Mixtures of Fluorescing Particles 223 9.3.4 Further Correlation Effects 224 9.3.5 Cross–Correlation Analysis 226 9.4 Optical Tweezers 230 9.4.1 Theoretical Background 230 9.4.2 Application Examples 236 9.4.2.1 Unfolding of RNA and DNA Hairpins 236 9.4.2.2 RNA Polymerase 238 9.4.2.3 DNA–Polymerase 239 9.5 Atomic Force Microscopy of Biomolecules 240 9.5.1 Principle of an AFM 241 9.5.2 Application Examples 242 9.5.2.1 Unfolding of DNA Hairpins 242 9.5.2.2 Receptor–Ligand Binding Forces 244 9.5.2.3 Protein Unfolding 244 9.6 Patch Clamping 245 9.6.1 Ion Channels 245 9.6.2 Patch Clamp Configurations 246 Problems 250 Bibliography 254 10 Ultrafast– and Nonlinear Spectroscopy 257 10.1 Introduction 257 10.2 Nonlinear Microscopy and Spectroscopy 258 10.2.1 Multiphoton Excitation 258 10.2.2 Advantages and Disadvantages of Two–Photon Excitation in Fluorescence Microscopy 259 10.2.3 How are Nonlinear Optical Signals Observed From Biological Samples? 262 10.2.4 Further Distinct Properties and Advantages of Two–Photon Excitation 263 10.2.5 Wavemixing and Other Nonlinear Optical Techniques 266 10.3 Ultrafast Spectroscopy 270 10.3.1 Pump–Probe Spectroscopy 270 10.3.2 Application Example: Ultrafast Light–Harvesting and Energy Conversion in Photosynthesis 274 10.3.2.1 Chlorophyll b®Chlorophyll a ® Reaction Centre Energy Flow 276 10.3.2.2 Carotenoid®Chlorophyll Energy Flow 278 Problems 280 Bibliography 282 11 DNA Sequencing and Next–Generation Sequencing Methods 285 11.1 Sanger Method 285 11.2 Next–Generation Sequencing Methods 287 11.2.1 Dye Sequencing (Approach I) 289 11.2.2 Sequencing by Ligation, Pyrosequencing and Ion Semiconductor Sequencing (Approaches II to IV) 292 11.2.2.1 Emulsion PCR 292 11.2.2.2 Sequencing by Ligation (Approach II) 294 11.2.2.3 Pyrosequencing (Approach III) 296 11.2.2.4 Ion Semiconductor Sequencing (Approach IV) 297 11.2.3 Single–Molecule Real–Time Sequencing (Approach V) 299 Problems 300 Bibliography 301 12 Special Techniques 303 12.1 Introduction 303 12.2 Fluorescing Nanoparticles 303 12.3 Surface Plasmon Resonance Detection 308 12.4 DNA Origami 310 12.5 DNA Microarrays 314 12.6 Flow Cytometry 317 12.7 Fluorescence In Situ Hybridization 319 12.8 Microspheres and Nanospheres 320 Problems 321 Bibliography 321 13 Assay Development, Readers and High–Throughput Screening 323 13.1 Introduction 323 13.2 Assay Development and Assay Quality 323 13.3 Microtitre Plates and Fluorescence Readers 326 13.4 Application Example: Drug Discovery and High–Throughput Screening 332 Problems 336 Bibliography 337 Index 339

Peter J. Walla is a group leader at the Max–Planck–Institute for Biophysical Chemistry in Göttingen (Germany), and Professor for Biophysical Chemistry at the University of Braunschweig. Having obtained his academic degrees from the Universities of Heidelberg and Göttingen, Professor Walla did postdoctoral research at the University of California at Berkeley, and headed a department in a Biotech company co–founded by Nobel Laureate Manfred Eigen, before taking up his current position.