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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
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.
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Analytical Techniques for the Elucidation of Protein Function
Edited by
Isao Suetake
Nakamura Gakuen University, Japan
Rohit K. Sharma
Panjab University, India
Hironobu Hojo
Institute for Protein Research, Osaka University, Japan
This edition first published 2023
© 2023 John Wiley & Sons Ltd
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Library of Congress Cataloging-in-Publication DataNames: Suetake, Isao, editor. | Sharma, Rohit K., editor. | Hojo, Hironobu, editor.Title: Analytical techniques for the elucidation of protein function / edited by Isao Suetake, Rohit K. Sharma, Hironobu Hojo.Description: Hoboken, NJ : John Wiley & Sons, 2023. | Includes bibliographical references and index.Identifiers: LCCN 2022038118 (print) | LCCN 2022038119 (ebook) | ISBN 9781119886327 (hardback) | ISBN 9781119886334 (pdf) | ISBN 9781119886341 (epub) | ISBN 9781119886358 (ebook)Subjects: LCSH: Proteins. | Electron paramagnetic resonance spectroscopy. | Nuclear magnetic resonance spectroscopy.
Classification: LCC QP551 .A485 2023 (print) | LCC QP551 (ebook) | DDC 572/.6--dc23/eng/20221005LC record available at https://lccn.loc.gov/2022038118LC ebook record available at https://lccn.loc.gov/2022038119
Cover Image: Courtesy of Tomohiro Hojo
Cover Design: Wiley
Set in 9.5/12.5pt STIXTwoText by Integra Software Services Pvt. Ltd, Pondicherry, India
Contents
Cover
Title page
Copyright
Preface
Editor’s Biographies
List of Contributors
1 EPR Spectroscopy
1.1 Outline of EPR Spectroscopy
1.1.1 Overview
1.2 Biological Applications of EPR
1.2.1 Proteins and Their Structures: Domain and Intrinsically Disordered Region
1.2.2 Introduction of Spin Probes on Proteins
1.2.3 Measurement of Constant Wave (CW)-EPR Spectrum
1.2.4 Application of CW-EPR to Protein (Clock Protein, Amyloid Proteins, and HP1)
1.2.4.1 Clock Proteins
1.2.4.2 Amyloid Proteins (Aβ Peptide, β2-microglobulin, α-synuclein, Tau, and Prion)
1.2.4.3 Heterochromatin Protein 1 (HP1)
1.2.5 Measurement of Longer Distance between Spin-spin (HP1, Tau, α-synuclein)
1.2.6 Biophysical Functions of Protein Dynamics
1.2.7 Summary/Conclusion
2 Introduction to Incoherent Neutron Scattering: A Powerful Technique to Investigate the Dynamics of Bio-macromolecules
2.1 Introduction
2.2 Basic Theory and Dynamical Information Obtained from iNS
2.2.1 Basic Principle of iNS Experiments
2.2.2 Incoherent Scattering Function
2.2.3 Dynamical Information Obtained by iNS
2.2.3.1 Elastic Incoherent Neutron Scattering (EINS)
2.2.3.2 Quasi-elastic Neutron Scattering (QENS)
2.3 Examples of Biological Applications of iNS
2.3.1 Dynamical Modulation of Proteins Caused by a Disease-causing Point Mutation
2.3.2 Dynamical Differences between Amyloid Polymorphic Fibrils Showing Different Levels of Cytotoxicity
2.3.3 New Theoretical Framework to Describe the Dynamical Behavior of Lipid Molecules
2.3.4 Separation of Dynamics of Protein-detergent Complexes
2.3.5 Hydration Water Mobility around Proteins
2.4 Summary
3 Elucidation of Protein Function Using Raman Spectroscopy
3.1 Introduction
3.2 Basic Principle and Working of Raman Spectroscopy
3.2.1 Theory and Frequencies of Raman Spectroscopy
3.2.2 Instrumentation
3.3 Advances in Raman Spectroscopy Techniques
3.3.1 Resonance Raman Spectroscopy for Protein Analysis
3.3.1.1 Ultraviolet Resonance Raman Spectroscopy
3.3.1.2 Time-resolved Resonance Raman Spectroscopy
3.3.2 Surface-enhanced Raman Spectroscopy (SERS)
3.3.3 Tip-enhanced Raman Spectroscopy
3.3.4 Polarized Raman Spectroscopy
3.3.5 Raman Crystallography
3.3.6 2D-COS Raman Spectroscopy
3.4 Applications
3.5 Conclusion
4 Fundamental Principles of Impedance Spectroscopy and its Biological Applications
4.1 Introduction
4.1.1 Basic Concept of Impedance Spectroscopy
4.1.2 Description of Impedance for Capacitors and Inductors
4.1.3 Nyquist Plot
4.1.4 Debye Model
4.1.5 Constant Phase and Warburg Element to Model Distorted and Diffusive Components
4.2 Biological Applications of Impedance Spectroscopy
4.2.1 Detection of DNA Hybridization and Photodamage
4.2.2 Detection and Analysis of Proteins
4.3 Conclusion
5 Mass Spectrometry Imaging
5.1 Introduction
5.2 Workflow of MSI
5.3 Mass Microscope
5.4 Visualization of Small Molecules (Pharmaceutical)
5.5 Structural Isomer Discrimination Imaging (Steroid Hormones)
5.6 Visualization of Proteins (Intact, Digestion)
5.7 Visualization of Protein Function (Enzymatic Activity Visualization)
5.8 Summary
6 Elucidation of Protein Function Using Single-molecule Monitoring by Quantum Dots
6.1 Introduction
6.1.1 Introduction to Quantum Dots
6.1.2 Types of Quantum Dots
6.1.2.1 Core Type QDs
6.1.2.2 Core/shell-type QDs
6.1.2.3 Alloyed-type QDs
6.2 Synthesis Methods
6.2.1 Wet-chemical Methods
6.2.2 Vapor-phase Methods
6.3 Bioconjugation
6.4 Analytical Methods for Single-molecule Monitoring by Quantum Dots
6.4.1 Epifluorescence Microscopy
6.4.2 Total Internal Reflection Fluorescence Microscope
6.4.3 Confocal Microscopy
6.4.4 pseudo-TIRFM
6.4.5 Single-point Edge Excitation Subdiffraction Microscopy
6.5 Applications
6.5.1 Application of Single-molecule Monitoring Using QD for Enlightening Nanoscale Neuroscience
6.5.2 Investigation of Diffusion Dynamics of Neuroreceptors in Cultured Neurons
6.5.3 Single-molecule Tracking of Neuroreceptors in Intact Brain Slices (in Vivo)
6.5.4 QD-tagged Neurotransmitter Transporters
6.5.5 QD Labeled Serotonin Transporter (SERT) to Understand Membrane Dynamics
6.5.6 Membrane Trafficking and Imaging of Dopamine Transporter (DAT) Using QDs
6.6 Limitations of QDs
6.7 Conclusion
7 Biological Solid-state NMR Spectroscopy
7.1 Introduction
7.2 Magnetic Interactions for NMR
7.2.1 Zeeman Interaction
7.2.2 Isotropic and Anisotropic Chemical Shifts
7.2.3 Homo- and Heteronuclear Dipolar Interactions
7.3 Methods for Solid-state NMR
7.3.1 Sample Preparation of Solid-state NMR
7.3.2 Experimental NMR Techniques for High-resolution Solid-state NMR
7.3.3 Fast MAS for 1H NMR
7.3.4 Multidimensional High-resolution NMR Experiments with Recoupling RF Pulse Sequences
7.3.5 Paramagnetic Effects for Structural Analysis
7.3.6 High-field DNP for Sensitivity Enhancement
7.3.7 Oriented Molecular Systems
7.4 Applications of Solid-state NMR to Biological Molecular Systems
7.4.1 Membrane Proteins and Peptides
7.4.2 Amyloid Fibrous Proteins
7.4.3 In-situ Cellular Biomolecules
7.5 Concluding Remarks
8 Electrically Induced Bubble Knife and Its Applications
8.1 Introduction
8.2 Electrically Induced Bubble Knife
8.3 Electrically Induced Bubble Injector
8.3.1 Bubble Formation with Reagent Interface
8.3.2 Simultaneous Injection and Ablation
8.4 Plasma-induced Bubble Injector
8.5 Protein Crystallization by Electrically Induced Bubbles
8.6 Protein Crystallization by Plasma-induced Bubbles
Index
End User License Agreement
List of Tables
CHAPTER 01
Table 1.2.1 Frequency and wavelength...
CHAPTER 06
Table 6.1 Various techniques...
CHAPTER 07
Table 7.1 Magnetic interactions...
Table 7.2 Proton and electron...
CHAPTER 08
Table 8.1 Classification...
Table 8.2 Experiment condition...
Table 8.3 Experiment condition...
List of Illustrations
CHAPTER 01
Figure 1.1.1 Energy levels...
Figure 1.1.2 Illustration...
Figure 1.1.3 Scale for the...
Figure 1.1.4 Coverage area...
Figure 1.1.5 Comparison of EPR...
Figure 1.1.6 (a) Recovery of the...
Figure 1.1.7 (a) A pulse sequence...
Figure 1.1.8 (a) Energy diagram...
Figure 1.1.9 ESEEM pattern...
Figure 1.1.10 Pulse sequences...
Figure 1.2.1 Peptide bond...
Figure 1.2.2 Molecular structures...
Figure 1.2.3 EPR spectrum...
Figure 1.2.4 Reaction of one...
Figure 1.2.5 Spin labeling of main...
Figure 1.2.6 Representative parameters...
Figure 1.2.7 Structure of KaiB...
Figure 1.2.8 Structure models...
Figure 1.2.9 Some modifications...
Figure 1.2.10 Schematic illustration...
CHAPTER 02
Figure 2.1 An illustration...
Figure 2.2 (a): A schematic...
Figure 2.3 Incoherent and...
Figure 2.4 Spatial distribution...
Figure 2.5 Simulation-based estimation...
Figure 2.6 A schematic illustration...
Figure 2.7 Atomic motions observed...
CHAPTER 03
Figure 3.1 (a) The region...
Figure 3.2 Representation...
Figure 3.3 Schematics of...
Figure 3.4 (a) TERS setup...
Figure 3.5 (a) The configuration...
Figure 3.6 RNAP active structure...
Figure 3.7 On-line and off-line...
Figure 3.8 Protein analysis...
CHAPTER 04
Figure 4.1 Overview of light...
Figure 4.2 Schematic concept...
Figure 4.3 Parameters to describe...
Figure 4.4 Schematic illustration...
Figure 4.5 Nyquist plots for simple...
Figure 4.6 Water molecules trying...
Figure 4.7 (a) Simulated real and...
Figure 4.8 Cole-Cole and Cole-Davidson...
Figure 4.9 Nyquist plots for model...
Figure 4.10 Schematic illustration...
Figure 4.11 Detection of DNA-hybridization...
Figure 4.12 Aggregated fibril structure...
Figure 4.13 Several experimental techniques...
Figure 4.14 Detection of Aβ oligomers...
CHAPTER 05
Figure 5.1 Mass spectrometry...
Figure 5.2 Schematic drawing...
Figure 5.3 An example of MSI...
Figure 5.4 Overview of reagents...
Figure 5.5 Structural isomer...
Figure 5.6 Example of tryptic-digested...
Figure 5.7 Concept of enzyme...
Figure 5.8 Evaluation of the...
Figure 5.9 ChAT activity imaging...
CHAPTER 06
Figure 6.1 (a) Varying band...
Figure 6.2 (a) The construction...
Figure 6.3 (a) Laser scanning...
Figure 6.4 (a) Spinning-disk...
Figure 6.5 (a) QD labeling of...
Figure 6.6 Time-lapse imaging...
CHAPTER 07
Figure 7.1 High-resolution...
Figure 7.2 RF pulse sequences...
Figure 7.3 DNP-NMR experiments...
Figure 7.4 (a) Two-dimensional...
Figure 7.5 (a) Core structure...
CHAPTER 08
Figure 8.1 Problems of electrical...
Figure 8.2 Process flow...
Figure 8.3 Characteristics...
Figure 8.4 Line of monodispersed...
Figure 8.5 Overview of electrical...
Figure 8.6 Input voltage...
Figure 8.7 Profiles of current...
Figure 8.8 Concept of cell ablation...
Figure 8.9 Ablation of cell...
Figure 8.10 Ablation of cell...
Figure 8.11 The concept of...
Figure 8.12 Ablation and...
Figure 8.13 Concept of bubble-plasma...
Figure 8.14 (a) Concept of the...
Figure 8.15 Electrical circuit...
Figure 8.16 Comparison of the protein...
Figure 8.17 Comparison of the average...
Figure 8.18 Comparison of the protein...
Figure 8.19 (a) Concept of protein...
Figure 8.20 Protein crystallization...
Figure 8.21 (a) Proposed microfluidic...
Figure 8.22 Production of protein crystals...
Figure 8.23 Identifying the hydrogen...
Guide
Cover
Title page
Copyright
Preface
Editor’s Biographies
List of Contributors
Table of Contents
Begin Reading
Index
End User License Agreement
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Preface
Proteins are fundamental biomolecules in living organisms and play various essential roles. They are composed of twenty amino acid connected by amide bonds and form secondary structures, such as α-helix and β-sheet, and ultimately, tertiary structures (also quaternary structures in the case of protein complexes). As the structure of proteins is very important for their function, various methods have been used to analyze them. The first structure clarified was myoglobin, which was reported in 1958 using X-ray crystallography. Nuclear magnetic resonance spectroscopy has also been highly advanced and widely used for protein structural analysis. Recently, cryoelectron microscopy has also become a powerful tool for this purpose. The structural data is deposited on the Protein Data Bank, and the number of solved structures is rapidly increasing. As the data is accessible from all over the world, it supports the progress of research on protein as well as the development of therapeutics.
Recently, the region in a protein that does not assume a particular three-dimensional structure, namely, the intrinsically disordered region (IDR), has been attracting much attention due to its importance for protein functions. IDR tends to form a liquid-liquid separated structure, which provides a site for various biological processes. IDR often receives various post-translational modifications, which are important to modify the function of proteins. In addition, the post-translational modifications often occur heterogeneously. To analyze the structure and function of these regions, the use of the above mentioned methods, such as X-ray crystallography and NMR, are not sufficient, as the regions are highly heterogeneous, mobile, and do not have firm tertiary structures. In addition, the analysis to identify the site and the kind of post-translational modification is required to elucidate how these modifications play roles.
In this book, we want to provide a brief introduction to less popular yet promising techniques to undertake functional and structural analyses of proteins, especially the intrinsically disordered region and the post-translational modifications. We anticipate that many of the readers are not familiar with the described techniques nor their theoretical background. Therefore, the authors describe each technique starting from a simple introduction and including a theoretical background, followed by the application of the method to the analysis of protein structure and function.
Chapter 1 describes the electron paramagnetic resonance spectroscopy technique. The method can be applied to the analysis of long-range interaction and fast motion of the proteins by specific spin labeling. Neutron scattering, described in Chapter 2, is an efficient method to analyze the dynamics of proteins. The method is particularly effective to analyze the dynamics of membrane proteins in lipid bilayers. Chapter 3 deals with Raman spectroscopy, which can clarify biological processes, such as protein-protein interaction and folding, by the analysis of scattering at the specific wave number indicative of a specific bond. This can be done with a small amount of sample without labeling. In Chapter 4, the author describes that the structure and aggregate formation of proteins can be globally analyzed by impedance measurement of a protein solution, which can be done without introducing any probes. Mass spectrometry imaging, described in Chapter 5, can analyze the distribution of various compounds in tissue samples with extreme high sensitivity using mass spectrometry. This method can be applied for the analysis of a wide range of compounds, as it identifies the target compounds by their molecular weight. Chapter 6 describes the single-molecule monitoring by quantum dots, which achieves the analysis of protein molecules one by one and is clearly advantageous when proteins with heterogeneous post-translational modifications are analyzed. Chapter 7 deals with solid-state NMR. The method can analyze proteins even in an aggregated state and micelles, which is very effective for the analysis of amyloid formation and membrane proteins in lipid bilayers. In Chapter 8, the novel method for the introduction of materials into cells as well as protein crystallization using bubble knife is described. These techniques are of great use for the analysis of proteins, which are difficult to be treated by conventional analytical methods.
We would like to express our sincere thanks to all the authors who contributed to this book. We also appreciate the efforts of reviewers who helped revise the text. Our thanks also go to Ms. Jenny Cossham and Ms. Elke Morice-Atkinson of John Wiley & Sons for their continuous help to realize the publication of this book.
We hope that the book will be a good introduction to the described techniques and contribute to increase their popularity for protein analysis, especially through research on the IDR. Also, we hope that the book will contribute to the further advance of protein science in the future.
Isao Suetake
Rohit K. Sharma
Hironobu Hojo
Editor’s Biographies
Isao Suetake, professor at the Graduate School of Nutritional Sciences, Nakamura Gakuen University, Japan.
Isao Suetake is a professor of nutrition at Nakamura Gakuen University. He graduated from the Faculty of Science, Osaka University, in 1990 with a bachelor’s degree and got his PhD in 1996 from the university. He specializes in biochemistry and molecular epigenetics and has researched the properties of enzymes responsible for DNA methylation and molecular mechanisms recognizing histone modifications. For more than ten years, he has collaborated with Prof. Hojo, who is one of editors of this book, to combine chemistry and bioscience for elucidating protein modifications.
Rohit K. Sharma, assistant professor at the Department of Chemistry, Panjab University, India.
Rohit K. Sharma obtained his PhD in medicinal chemistry, National Institute of Pharmaceutical Education and Research (NIPER) in 2009. His thesis was on the design and synthesis of antimicrobial peptides. In 2007, he underwent a research sojourn in the Helmholtz Centre for Infection Research (HZI), Germany. In 2009, he moved to Nanyang Technological University, Singapore, as a postdoctoral research fellow. He moved to his present position in Panjab University in 2011, where his research interests include understanding nano-peptide conjugates and self-assembling organic networks with applications in drug delivery and bio-sensing.
Hironobu Hojo, professor at the Institute for Protein Research, Osaka University, Japan.
Hironobu Hojo obtained his PhD in organic chemistry from Osaka University in 1994. His thesis was on the development of the method for chemical protein synthesis, the thioester method. In 1998, he moved to Tokai University and started to extend the thioester method for glycoprotein synthesis. He moved to his present position, professor at the Institute for Protein Research, Osaka University, in 2013 and is developing a chemical approach toward the understanding of the function of post-translationally modified proteins.
List of Contributors
Toshimichi FujiwaraOsaka UniversitySuita, OsakaJapan
Hironobu HojoOsaka UniversitySuita, OsakaJapan
Gurpreet K. SoniPanjab UniversityChandigarhIndia
Alisha LalhallPanjab UniversityChandigarhIndia
Saima MalikPanjab UniversityChandigarhIndia
Tatsuhito MatsuoUniversité Grenoble AlpesGrenobleFrance
Hiroyuki MinoNagoya UniversityNagoyaJapan
Judith PetersUniversité Grenoble AlpesGrenobleFrance
Deepika SharmaPanjab UniversityChandigarhIndia
Isao SuetakeNakamura Gakuen UniversityFukuokaJapan
Rohit K. SharmaPanjab UniversityChandigarhIndia
Shuichi ShimmaOsaka UniversitySuita, OsakaJapan
Maitrayee U. TrivediPanjab UniversityChandigarhIndia
Yusuke TsutsuiKyoto UniversityKyotoJapan
Nishima WangooPanjab UniversityChandigarhIndia
Yoko YamanishiKyushu UniversityFukuokaJapan
1.2 Biological Applications of EPR
Isao Suetake1,2, Risa Mutoh3, Yuichi Mishima2, Masatomo So2, and Hironobu Hojo2,*
1 Department of Nutritional Sciences, Faculty of Nutritional Sciences, Nakamura Gakuen University, Fukuoka, Japan2 Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka, Japan3 Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba, Japan* Corresponding author
1.2.1 Proteins and Their Structures: Domain and Intrinsically Disordered Region
Proteins are generally composed of 20 amino acids. In proteins, amino acids are connected by peptide bonds (Figure 1.2.1). The polypeptide backbone is composed of NH, carbonyl (CO), and a CHR group, in which R is the side chain of amino acids. The side chain is a specific group to individual amino acids. The sequence of amino acids in a polypeptide chain is the simplest level of protein structure, the primary structure. The locally folded structures, in which the interactions between atoms of the polypeptide backbone occur, are called the secondary structure. The most common types of secondary structures are the α-helix and the β-sheet. Both structures are held in shape by hydrogen bonds, which are formed between the carbonyl oxygen of one amino acid and the amido hydrogen of another. By associating the secondary structures, functional domains are formed. The three-dimensional domain structure has been elucidated by X-ray diffraction experiments on crystals and NMR in solution. The structures have been collected in an easily accessible protein data bank (PDB). More than one-third of proteins in eukaryotic cells, however, have disordered regions that are more than 30 amino acids in length [1]. Within disordered regions, charged amino acids are frequently observed, while the hydrophobic amino acids are rare. The flexibility of these regions allows conformational change and interaction with multiple partners. EPR is an excellent technique to understand the dynamics and weak interactions.
Figure 1.2.1 Peptide bond structure. A peptide is made up of an amino acid connected by peptide bonds. R (1–3) indicates side chain of amino acid. Phi (φ) and psi (ψ) dihedral angle rotations of the amino acid are shown.
