Modern Biophysical Chemistry E-Book

Peter J. Walla

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 551

Veröffentlichungsjahr: 2014

Beschreibung

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

Leseprobe

CONTENTS

Cover

Related Titles

Title Page

Copyright

Dedication

Foreword to the Second Edition

Introduction

Part One: Basic Methods in Biophysical Chemistry

Chapter 1: Basic Optical Principles

1.1 Introduction

1.2 What Does the Electronic Structure of Molecules Look Like? Orbitals, Wave Functions and Bonding Interactions

1.3 How Does Light Interact with Molecules? Transition Densities and the Transition Dipole Moment

1.4 Absorption Spectra of Molecules in Liquid Environments. Vibrational Excitation and the Franck–Condon Principle

1.5 What Happens After Molecules have Absorbed Light? Fluorescence, Nonradiative Transitions and the Triplet State

1.6 Quantitative Description of all Processes: Quantum Efficiencies, Kinetics of Excited State Populations and the Jablonski Diagram

Bibliography

Chapter 2: Optical Properties of Biomolecules

2.1 Introduction

2.2 Experimental Determination of Absorption and Fluorescence Spectra

2.3 Optical Properties of Proteins and DNA

2.4 Optical Properties of Important Cofactors

Bibliography

Chapter 3: Basic Fluorescence Techniques

3.1 Introduction

3.2 Fluorescent Labelling and Linking Techniques

3.3 Fluorescence Detection Techniques

3.4 Fluorescence Polarization Anisotropy

3.5 Förster Resonance Energy Transfer

3.6 Fluorescence Kinetics

3.7 Fluorescence Recovery after Photobleaching

3.8 Biochemiluminescence

Bibliography

Chapter 4: Chiroptical and Scattering Methods

4.1 Chiroptical Methods

4.2 Light Scattering

4.3 Vibrational Spectra of Biomolecules

Bibliography

Chapter 5: Magnetic Resonance Techniques

5.1 Nuclear Magnetic Resonance of Biomolecules

5.2 Electron Paramagnetic Resonance

Bibliography

Chapter 6: Mass Spectrometry

6.1 Introduction

6.2 MALDI-TOF

6.3 ESI-MS

6.4 Structural and Sequence Analysis Using Mass Spectrometry

Bibliography

Part Two: Advanced Methods in Biophysical Chemistry

Chapter 7: Fluorescence Microscopy

7.1 Introduction

7.2 Conventional Fluorescence Microscopy

7.3 Total Internal Reflection Fluorescence Microscopy

7.4 Light-Sheet Microscopy

Bibliography

Chapter 8: Super-Resolution Fluorescence Microscopy

8.1 Stimulated Emission Depletion (STED) Microscopy

8.2 Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM)

8.3 3D Super-Resolution Fluorescence Microscopy

8.4 Imaging of Live Cells

8.5 Multicolour Super-Resolution Fluorescence Microscopy

8.6 Structured Illumination Microscopy

8.7 SOFI

8.8 Final Comparison

Bibliography

Chapter 9: Single-Biomolecule Techniques

9.1 Introduction

9.2 Optical Single-Molecule Detection

9.3 Fluorescence Correlation Spectroscopy

9.4 Optical Tweezers

9.5 Atomic Force Microscopy of Biomolecules

9.6 Patch Clamping

Bibliography

Chapter 10: Ultrafast- and Nonlinear Spectroscopy

10.1 Introduction

10.2 Nonlinear Microscopy and Spectroscopy

10.3 Ultrafast Spectroscopy

Bibliography

Chapter 11: DNA Sequencing and Next-Generation Sequencing Methods

11.1 Sanger Method

11.2 Next-Generation Sequencing Methods

Bibliography

Chapter 12: Special Techniques

12.1 Introduction

12.2 Fluorescing Nanoparticles

12.3 Surface Plasmon Resonance Detection

12.4 DNA Origami

12.5 DNA Microarrays

12.6 Flow Cytometry

12.7 Fluorescence In Situ Hybridization

12.8 Microspheres and Nanospheres

Chapter 13: Assay Development, Readers and High-Throughput Screening

13.1 Introduction

13.2 Assay Development and Assay Quality

13.3 Microtitre Plates and Fluorescence Readers

13.4 Application Example: Drug Discovery and High-Throughput Screening

Bibliography

Index

End User License Agreement

List of Tables

Table 2.1

Table 2.2

Table 3.1

Table 4.1

Table 4.2

Table 6.1

Table 9.1

Table 10.1

List of Illustrations

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 1.9

Figure 1.10

Figure 1.11

Figure 1.12

Figure 1.13

Figure 1.14

Figure 1.15

Figure 1.16

Figure 1.17

Figure 1.18

Figure 1.19

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.11

Figure 2.12

Figure 2.13

Figure 2.14

Figure 2.15

Figure 2.16

Figure 2.17

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 3.16

Figure 3.17

Figure 3.18

Figure 3.19

Figure 3.20

Figure 3.21

Figure 3.22

Figure 3.23

Figure 3.24

Figure 3.25

Figure 3.26

Figure 3.27

Figure 3.28

Figure 3.29

Figure 3.30

Figure 3.31

Figure 3.32

Figure 3.33

Figure 3.34

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 5.12

Figure 5.13

Figure 5.14

Figure 5.15

Figure 5.16

Figure 5.17

Figure 5.18

Figure 5.19

Figure 5.20

Figure 5.21

Figure 5.22

Figure 5.23

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

Figure 6.9

Figure 6.10

Figure 6.11

Figure 6.12

Figure 6.13

Figure 6.14

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 7.9

Figure 7.10

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Figure 8.5

Figure 8.6

Figure 8.7

Figure 8.8

Figure 8.9

Figure 8.10

Figure 8.11

Figure 8.12

Figure 9.1

Figure 9.2

Figure 9.3

Figure 9.4

Figure 9.5

Figure 9.6

Figure 9.7

Figure 9.8

Figure 9.9

Figure 9.10

Figure 9.11

Figure 9.12

Figure 9.13

Figure 9.14

Figure 9.15

Figure 9.16

Figure 9.17

Figure 9.18

Figure 9.19

Figure 9.20

Figure 9.21

Figure 9.22

Figure 9.23

Figure 9.24

Figure 9.25

Figure 9.26

Figure 9.27

Figure 9.28

Figure 9.29

Figure 9.30

Figure 9.31

Figure 9.32

Figure 9.33

Figure 9.34

Figure 9.35

Figure 9.36

Figure 10.1

Figure 10.2

Figure 10.3

Figure 10.4

Figure 10.5

Figure 10.6

Figure 10.7

Figure 10.8

Figure 10.9

Figure 10.10

Figure 10.11

Figure 10.12

Figure 10.13

Figure 10.14

Figure 10.15

Figure 10.16

Figure 10.17

Figure 10.18

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 11.6

Figure 11.7

Figure 11.8

Figure 11.9

Figure 11.10

Figure 11.11

Figure 12.1

Figure 12.2

Figure 12.3

Figure 12.4

Figure 12.5

Figure 12.6

Figure 12.7

Figure 12.8

Figure 12.9

Figure 12.10

Figure 12.11

Figure 12.12

Figure 12.13

Figure 12.14

Figure 12.15

Figure 12.16

Figure 12.17

Figure 13.1

Figure 13.2

Figure 13.3

Figure 13.4

Figure 13.5

Figure 13.6

Figure 13.7

Figure 13.8

Figure 13.9

Figure 13.10

Figure 13.11

Guide

Cover

Table of Contents

Begin Reading

Begin Reading

Part 1

Chapter 1

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Modern Biophysical Chemistry

Detection and Analysis of Biomolecules

Second, Updated and Expanded Edition

Peter Jomo Walla

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.

© 2014 Wiley-VCH Verlag GmbH # Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

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Dedication

To my wonderful wife Uli and my great kids Christian, Maike, Paul and Johanna

Foreword to the Second Edition

It has now been five years since the first edition of this book entitled “Modern Biophysical Chemistry” appeared and of course it was high time that this book was updated with the latest exciting developments that are now established enough to be included in a text book. As already mentioned in the first edition, the field of biophysical chemistry is huge, covering aspects of chemistry, biology, physics and even medicine and so it is not easy to assess which aspects are really the most important ones that must be included. As for the first edition, I have tried to make a selection of methods and application examples that contain general concepts that also cover the basis for most of the techniques and applications that are not considered explicitly in the book. Since this selection can be done in many different ways I ask those who are disappointed that their method or application is not explicitly included here to excuse me. The goal was a book that allows a comparatively quick insight to be gained into the very large range of possibilities provided by modern biophysical chemistry, while still being detailed enough to use this knowledge for the first steps in actually applying it for research.

Amongst the important developments of recent years are certainly significant improvements achieved in DNA sequencing by next-generation methods as well as super-resolution microscopy that goes beyond the resolution of conventional, diffraction-limited microscopy. Therefore, these two developments are now represented by two, entirely new chapters in the book. Also, other new developments have been included, such as light-sheet microscopy, introduced now in Chapter 7, and DNA origami techniques in Chapter 12. In addition, new problems have been added. To further improve the clarity many figures have been coloured and, in general, we have made major efforts to optimize the clarity and conciseness throughout the entire book. To help readers focus on the important equations in all mathematical subjects they are now marked by black boxes throughout the entire book.

I would like to thank again all persons who helped me to improve the first edition and of course also those who helped me with the second edition with their very useful and often essential comments.

Special thanks go to Dr Stefan Bode, Dr Anna Cypionka, Professor Dr Christian Eggeling, Professor Dr Jörg Enderlein, Dr Jan Frähmcke, Professor Dr Karl-Heinz Gericke, Matthias Grunwald, Dr Ulrich Haupts, Dr Hendrik Hippchen, Professor Dr Henrike Heise, Christoph-Peter Holleboom, Chao-Chen Lin, Dr Martin Michels, Professor Dr Filipp Oesterhelt, Dr Wiebke Pohl, Professor Dr Christoph Schmidt, Sabrina Schröder, Professor Dr Jakob Sørensen, Professor Dr Dirk Schwarzer, Dr Michael Teufel, Dr Andreas Volkmer and finally Laura van den Heuvel. In addition, I especially thank Silke Lubahn for generating most of the figures in this book and Julia Lüttich for her extremely valuable support throughout the entire time this second edition was being prepared. Gratitude is also expressed to Dr Pen-Nan Liao for his assistance with the solutions to the questions asked in the book. Last, but not least, I would like to thank all persons at Wiley-VCH who helped with the production of this book and especially Lesley Fenske, Dr Peter Capper, Mamta Pujari and Dr Frank Weinreich for their patience regarding my special requests and wishes.

Braunschweig and Göttingen, May 2014

Introduction

What is Biophysical Chemistry? – An Example from Drug Screening

Biophysical chemistry is a fascinating field of research because it combines aspects of chemistry, biology, physics and sometimes even medicine in one discipline. Owing to this diversity it is difficult to give an exact definition of biophysical chemistry. In principle, everything in biology or medicine is based on a chemical or physical foundation. For a physical chemist, one reasonable definition is ‘Biophysical chemistry is the application of principles known from physical chemistry to elucidate biomolecular and biochemical questions’. For a biologist a reasonable definition might be ‘Biophysical chemistry is the description of the physicochemical properties of biomolecules’.

Actually, it makes a lot more sense to answer the question ‘Why do we need biophysical chemistry?’. In recent years more and more questions relevant to biology have been answered using methods originating from the field of physics or physical chemistry. These problems require at least some basic understanding in all three disciplines. However, often a physicist or chemist feels uncomfortable talking about topics that seem to be quite simple for a biologist and vice versa. In many cases it turns out that something that sounded very complicated to one scientist is not difficult at all after he or she realizes that the other scientist is simply using unfamiliar wording. An example is the definition of a ‘vector’. Chemists and physicists usually regard a vector as a mathematical object. However, if molecular biologists are talking about vectors they often mean a plasmid vector for transferring genetic material into a cell. The field of biophysical chemistry is a bridge between these disciplines. The following example illustrates a typical problem that can only be solved with a basic knowledge of all these disciplines.

For the development of a drug, in pharmaceutical research in many cases one very important parameter is the affinity of potential drug candidates for a specific receptor or enzyme. The mechanism by which many drugs act is simply based on their ability to selectively block the active site of specific biomolecules. For example, the biomolecular targets of many antibiotics are enzymes responsible for the cell-wall synthesis of bacteria. Since it is very hard to find such compounds that also have as few side effects as possible it is useful to look at as many compound structures as possible. Pharmaceutical companies often have a very large pool – up to millions – of already synthesized compound structures. Often, in a first step in the process of industrial drug development, many of these compound structures are tested for their affinity to a specific target using high-throughput screening (HTS). If a compound structure with a high affinity can be found (a ‘Hit’) it can be used as starting point for further drug development. But how can the affinity of a million compounds be measured with sufficient speed and accuracy? A day lasts 86 400 s. If the accurate measurement of the binding affinity takes only one second per compound, then more than 11 days of constant measurements are required for one million compounds.