ANALYTICAL TECHNIQUES FOR THE ELUCIDATION OF PROTEIN FUNCTION
An essential aid for scientists seeking alternative techniques for investigating proteins
Proteins are the building blocks of living organisms, and they play an enormous range of fundamental roles in sustaining and shaping life. The critical determinant of a protein’s function is its structure, and the analysis of protein structures has therefore become a significant component of biological research. In recent years, longstanding analytical techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have been supplemented by a number of new methods which promise to revolutionize the study of proteins and their functions.
Analytical Techniques for the Elucidation of Protein Function serves as an introduction to these techniques, which are especially crucial for analyzing intrinsically disordered regions and post-translational modifications. These have revolutionized the study of proteins in recent years, and conventional methods for analyzing protein structures are no longer sufficient to work through their ramifications. This book therefore brings greater awareness of techniques which promise to produce the very cutting edge of protein research.
Analytical Techniques for the Elucidation of Protein Function readers will find:
- A discussion of techniques including electron paramagnetic resonance (ESR) spectroscopy, neutron scattering, Raman imaging, and more
- Both theoretical background and practical applications for each technique
- Contributions from leading international researchers into protein structure and function
This practically focused text is a valuable reference for protein and peptide analysis and synthesis researchers, as well as for graduate and advanced undergraduate students in the life sciences.
Table of Contents
Preface ix
Editor’s Biographies xi
List of Contributors xiii
1 EPR Spectroscopy 1
1.1 Outline of EPR Spectroscopy 2
Hiroyuki Mino
1.1.1 Overview 2
1.2 Biological Applications of EPR 13
Isao Suetake, Risa Mutoh, Yuichi Mishima, Masatomo So, and Hironobu Hojo
1.2.1 Proteins and Their Structures: Domain and Intrinsically Disordered Region 13
1.2.2 Introduction of Spin Probes on Proteins 14
1.2.3 Measurement of Constant Wave (CW)-EPR Spectrum 19
1.2.4 Application of CW-EPR to Protein (Clock Protein, Amyloid Proteins, and HP1) 21
1.2.4.1 Clock Proteins 22
1.2.4.2 Amyloid Proteins (Aβ Peptide, β 2 -microglobulin, α-synuclein, Tau, and Prion) 23
1.2.4.3 Heterochromatin Protein 1 (HP1) 26
1.2.5 Measurement of Longer Distance between Spin-spin (HP1, Tau, α-synuclein) 29
1.2.6 Biophysical Functions of Protein Dynamics 31
1.2.7 Summary/Conclusion 31
2 Introduction to Incoherent Neutron Scattering: A Powerful Technique to Investigate the Dynamics of Bio-macromolecules 39
Tatsuhito Matsuo and Judith Peters
2.1 Introduction 39
2.2 Basic Theory and Dynamical Information Obtained from iNS 42
2.2.1 Basic Principle of iNS Experiments 42
2.2.2 Incoherent Scattering Function 45
2.2.3 Dynamical Information Obtained by iNS 51
2.2.3.1 Elastic Incoherent Neutron Scattering (EINS) 52
2.2.3.2 Quasi-elastic Neutron Scattering (QENS) 53
2.3 Examples of Biological Applications of iNS 58
2.3.1 Dynamical Modulation of Proteins Caused by a Disease-causing Point Mutation 58
2.3.2 Dynamical Differences between Amyloid Polymorphic Fibrils Showing Different Levels of Cytotoxicity 59
2.3.3 New Theoretical Framework to Describe the Dynamical Behavior of Lipid Molecules 60
2.3.4 Separation of Dynamics of Protein-detergent Complexes 61
2.3.5 Hydration Water Mobility around Proteins 62
2.4 Summary 63
3 Elucidation of Protein Function Using Raman Spectroscopy 69
Saima Malik, Maitrayee U. Trivedi, Gurpreet K. Soni, and Rohit K. Sharma
3.1 Introduction 69
3.2 Basic Principle and Working of Raman Spectroscopy 71
3.2.1 Theory and Frequencies of Raman Spectroscopy 71
3.2.2 Instrumentation 73
3.3 Advances in Raman Spectroscopy Techniques 74
3.3.1 Resonance Raman Spectroscopy for Protein Analysis 74
3.3.1.1 Ultraviolet Resonance Raman Spectroscopy 75
3.3.1.2 Time-resolved Resonance Raman Spectroscopy 76
3.3.2 Surface-enhanced Raman Spectroscopy (SERS) 77
3.3.3 Tip-enhanced Raman Spectroscopy 80
3.3.4 Polarized Raman Spectroscopy 83
3.3.5 Raman Crystallography 85
3.3.6 2D-COS Raman Spectroscopy 88
3.4 Applications 91
3.5 Conclusion 92
4 Fundamental Principles of Impedance Spectroscopy and its Biological Applications 101
Yusuke Tsutsui
4.1 Introduction 101
4.1.1 Basic Concept of Impedance Spectroscopy 101
4.1.2 Description of Impedance for Capacitors and Inductors 105
4.1.3 Nyquist Plot 106
4.1.4 Debye Model 108
4.1.5 Constant Phase and Warburg Element to Model Distorted and Diffusive Components 111
4.2 Biological Applications of Impedance Spectroscopy 113
4.2.1 Detection of DNA Hybridization and Photodamage 113
4.2.2 Detection and Analysis of Proteins 115
4.3 Conclusion 119
5 Mass Spectrometry Imaging 125
Shuichi Shimma
5.1 Introduction 125
5.2 Workflow of MSI 126
5.3 Mass Microscope 128
5.4 Visualization of Small Molecules (Pharmaceutical) 128
5.5 Structural Isomer Discrimination Imaging (Steroid Hormones) 130
5.6 Visualization of Proteins (Intact, Digestion) 133
5.7 Visualization of Protein Function (Enzymatic Activity Visualization) 134
5.8 Summary 139
6 Elucidation of Protein Function Using Single-molecule Monitoring by Quantum Dots 143
Maitrayee U. Trivedi, Deepika Sharma, Alisha Lalhall, Rohit K. Sharma, and Nishima Wangoo
6.1 Introduction 143
6.1.1 Introduction to Quantum Dots 144
6.1.2 Types of Quantum Dots 145
6.1.2.1 Core Type QDs 145
6.1.2.2 Core/shell-type QDs 147
6.1.2.3 Alloyed-type QDs 148
6.2 Synthesis Methods 148
6.2.1 Wet-chemical Methods 150
6.2.2 Vapor-phase Methods 150
6.3 Bioconjugation 151
6.4 Analytical Methods for Single-molecule Monitoring by Quantum Dots 152
6.4.1 Epifluorescence Microscopy 152
6.4.2 Total Internal Reflection Fluorescence Microscope 153
6.4.3 Confocal Microscopy 154
6.4.4 pseudo-TIRFM 154
6.4.5 Single-point Edge Excitation Subdiffraction Microscopy 156
6.5 Applications 156
6.5.1 Application of Single-molecule Monitoring Using QD for Enlightening Nanoscale Neuroscience 156
6.5.2 Investigation of Diffusion Dynamics of Neuroreceptors in Cultured Neurons 157
6.5.3 Single-molecule Tracking of Neuroreceptors in Intact Brain Slices (in Vivo) 158
6.5.4 QD-tagged Neurotransmitter Transporters 160
6.5.5 QD Labeled Serotonin Transporter (SERT) to Understand Membrane Dynamics 160
6.5.6 Membrane Trafficking and Imaging of Dopamine Transporter (DAT) Using QDs 161
6.6 Limitations of QDs 163
6.7 Conclusion 163
7 Biological Solid-state NMR Spectroscopy 169
Toshimichi Fujiwara
7.1 Introduction 169
7.2 Magnetic Interactions for NMR 170
7.2.1 Zeeman Interaction 170
7.2.2 Isotropic and Anisotropic Chemical Shifts 170
7.2.3 Homo- and Heteronuclear Dipolar Interactions 171
7.3 Methods for Solid-state NMR 173
7.3.1 Sample Preparation of Solid-state NMR 173
7.3.2 Experimental NMR Techniques for High-resolution Solid-state NMR 174
7.3.3 Fast MAS for 1 H NMR 176
7.3.4 Multidimensional High-resolution NMR Experiments with Recoupling RF Pulse Sequences 176
7.3.5 Paramagnetic Effects for Structural Analysis 177
7.3.6 High-field DNP for Sensitivity Enhancement 178
7.3.7 Oriented Molecular Systems 179
7.4 Applications of Solid-state NMR to Biological Molecular Systems 180
7.4.1 Membrane Proteins and Peptides 180
7.4.2 Amyloid Fibrous Proteins 182
7.4.3 In-situ Cellular Biomolecules 184
7.5 Concluding Remarks 184
8 Electrically Induced Bubble Knife and Its Applications 191
Yoko Yamanishi
8.1 Introduction 191
8.2 Electrically Induced Bubble Knife 192
8.3 Electrically Induced Bubble Injector 199
8.3.1 Bubble Formation with Reagent Interface 200
8.3.2 Simultaneous Injection and Ablation 200
8.4 Plasma-induced Bubble Injector 201
8.5 Protein Crystallization by Electrically Induced Bubbles 202
8.6 Protein Crystallization by Plasma-induced Bubbles 207
Index 215
