Advances in Protein Molecular and Structural Biology Methods

Advances in Protein Molecular and Structural Biology Methods offers a complete overview of the latest tools and methods applicable to the study of proteins at the molecular and structural level. The book begins with sections exploring tools to optimize recombinant protein expression and biophysical techniques such as fluorescence spectroscopy, NMR, mass spectrometry, cryo-electron microscopy, and X-ray crystallography. It then moves towards computational approaches, considering structural bioinformatics, molecular dynamics simulations, and deep machine learning technologies. The book also covers methods applied to intrinsically disordered proteins (IDPs)followed by chapters on protein interaction networks, protein function, and protein design and engineering.

It provides researchers with an extensive toolkit of methods and techniques to draw from when conducting their own experimental work, taking them from foundational concepts to practical application.

Cover image
Title page
Table of Contents
Copyright
Dedicated to
Contributors
About the Editors
Foreword
Preface
Chapter 1: Strategies to improve the expression and solubility of recombinant proteins in E. coli
Abstract
1: Introduction
2: Before starting with protein expression
3: Materials required
4: Standard protocol for recombinant protein expression in E. coli
5: Troubleshooting strategies
6: Conclusion and future perspectives
Chapter 2: Advances in heterologous protein expression strategies in yeast and insect systems
Abstract
Acknowledgment
1: Introduction
2: Heterologous protein expression strategies in yeast systems
3: Heterologous protein expression strategies in insect systems
4: Conclusion of baculovirus expression systems
Chapter 3: Methods for transient expression and purification of monoclonal antibodies in mammalian cells
Abstract
1: Introduction
2: Background experimental preparation
3: Materials required for antibody purification
4: Detailed step-by-step protocol for antibody purification
5: Troubleshooting problems
6: Conclusions
Chapter 4: Methods for recombinant production and purification of intrinsically disordered proteins
Abstract
1: Before you begin
2: Materials and equipment
3: Step-by-step method details
4: Expected outcomes
5: Optimization and troubleshooting
Chapter 5: Methods to determine the oligomeric structure of proteins
Abstract
1: Introduction
2: Electrophoretic methods
3: Size exclusion chromatography
4: Dynamic light scattering
5: Circular dichroism spectroscopy
6: Fluorescence-based methods
7: Analytical ultracentrifugation
8: X-ray crystallography and NMR spectroscopy
9: Mass spectroscopy
10: Atomic force microscopy
11: Co-immunoprecipitation
12: Computational methods
13: Conclusions
Chapter 6: Multimodal methods to study protein aggregation and fibrillation
Abstract
Acknowledgments
1: Introduction
2: Combination of in vitro techniques to evaluate isolated protein aggregates and fibrils
3: Multimodal methods to evaluate the aggregation of proteins in cells tissues and living system
Chapter 7: Experimental methods to study the thermodynamics of protein–protein interactions
Abstract
1: Introduction
2: Criteria for forming a protein–protein interaction
3: Characteristic features of PPI interfaces
4: Thermodynamic parameters associated with PPI
5: Techniques to study the thermodynamics of PPI
6: Conclusions
Chapter 8: Experimental methods to study the kinetics of protein–protein interactions
Abstract
Acknowledgment
1: Introduction
2: Surface plasmon resonance
3: Bio-layer interferometry
4: Microscale thermophoresis
5: Isothermal titration calorimetry
6: Quartz crystal microbalance
7: Conclusions
Chapter 9: Computational techniques for studying protein-protein interactions
Abstract
1: Introduction
2: Types of protein-protein complexes
3: PPIs as targets for drug discovery
4: Mining PPIs
5: Computational techniques in PPI detection
6: Comparison of available computational approaches
7: PPI databases
8: PPI network and visualization
9: Conclusion and future perspectives
Chapter 10: Experimental methods to study protein–nucleic acid interactions
Abstract
1: Introduction
2: Single-molecule approaches for the identification and validation of protein–nucleic acid interactions
3: Investigation of protein–nucleic acid interactions in mammalian cell lines
4: Conclusions
Chapter 11: Advanced computational tools for quantitative analysis of protein–nucleic acid interfaces
Abstract
Acknowledgment
1: Protein–RNA complexes
2: Datasets for studying protein–RNA interfaces
3: Tools for the analysis of protein–RNA interfaces
4: Tools for protein–RNA binding site prediction
5: Tools for protein–RNA binding affinity prediction
6: Tools for hot spots at protein–RNA interfaces
7: A brief survey of tools for studying protein–DNA interactions
8: Conclusions
Chapter 12: Experimental techniques to study protein dynamics and conformations
Abstract
1: Introduction
2: Various methods to study protein dynamics and conformations
3: Conclusion and future perspectives
Chapter 13: Computational techniques to study protein dynamics and conformations
Abstract
Acknowledgments
1: Introduction
2: “Realistic” methods: Molecular dynamics and enhanced sampling
3: “Simplified” approaches: Coarse-graining and path-sampling algorithms
4: A case study: The open-to-close transition of the ribose-binding protein
5: Summary and conclusions
Chapter 14: Application of circular dichroism spectroscopy in studying protein folding, stability, and interaction
Abstract
1: Introduction
2: Theory of circular dichroism
3: Application of CD spectroscopy
4: Time-resolved CD spectroscopy and its uses in protein folding kinetics
5: Conclusion and future perspectives
Conflict of interests
Chapter 15: Studying protein-folding dynamics using single-molecule fluorescence methods
Abstract
1: Introduction
2: Single-molecule fluorescence techniques for protein-folding dynamics
3: Conclusion and future perspectives
Chapter 16: Advances in liquid-state NMR spectroscopy to study the structure, function, and dynamics of biomacromolecules
Abstract
1: Introduction to liquid-state NMR spectroscopy
2: Liquid-state NMR spectroscopy of biomacromolecules
3: Biomolecular behavior and drug discovery
4: NMR of biomacromolecules in living cells
5: Summary
Chapter 17: In-cell NMR spectroscopy: A tool to study cellular structure biology
Abstract
1: Introduction
2: Overview of in-cell NMR
3: Bioreactor systems for in-cell NMR observations
4: Applications of in-cell NMR
5: Conclusion and future perspectives
Chapter 18: Current trends in membrane protein crystallography
Abstract
Acknowledgments
1: Introduction
2: Expression screening of membrane proteins for crystallization
3: Detergent screening and fluorescence size exclusion chromatography of membrane proteins
4: Crystallization of membrane proteins
5: Engineering membrane proteins to facilitate crystal formation
6: X-ray sources
7: Detectors
8: Time-resolved crystallography
9: X-ray free-electron laser
10: Conclusion and future perspectives
Chapter 19: Advances in sample preparation and data processing for single-particle cryo-electron microscopy
Abstract
1: Introduction
2: Sample quality is the key to high-resolution structure determination
3: Grid preparation for SPA
4: Time-resolved cryoEM
5: Advances in SPA data collection and processing
6: AI/ML-based approaches in cryoEM data processing pipeline
7: Conclusion and future perspectives
Chapter 20: Advanced mass spectrometry-based methods for protein molecular-structural biologists
Abstract
Acknowledgments
1: Introduction
2: Data-independent acquisitions (DIA) for accurate protein quantification
3: Resolving protein structures using DIA-MS
4: Conclusions and outlooks
Chapter 21: Developments, advancements, and contributions of mass spectrometry in omics technologies
Abstract
1: Introduction
2: Omics mass spectrometry
3: Mass spectrometry in Omics technologies
4: Recent developments in the mass spectrometer
5: Fragmentation principles
6: Conclusion and future perspectives
Chapter 22: Role of structural biology methods in drug discovery
Abstract
1: Introduction
2: Structural biology aided selection of drug targets
3: Role of experimental and computational approaches in drug discovery
4: Virtual screening
5: Enhancement of ligand specificity
6: Optimization of hits and drug-likeness
7: Development of peptidomimetics
8: Conclusion and future perspectives
Chapter 23: Prediction, validation, and analysis of protein structures: A beginner’s guide
Abstract
1: Introduction
2: Protein structure modeling
3: Protein structure refinement and validation
4: Protein structure analysis and importance of protein folding
5: Recent advances in in silico protein structure determination
6: Conclusion and future perspectives
Chapter 24: Advances in structure-based virtual screening for drug discovery
Abstract
1: Introduction
2: Drug design and the computers
3: Conclusion and future perspectives
Chapter 25: Methods and applications of machine learning in structure-based drug discovery
Abstract
Acknowledgments
1: Introduction
2: Protein crystallography and AI-assisted drug discovery
3: Application of ML in protein structure prediction (in silico approach)
4: Virtual screening
5: Conclusion and future perspectives
Chapter 26: Molecular dynamics simulations: Principles, methods, and applications in protein conformational dynamics
Abstract
Acknowledgments
1: Introduction
2: Applications of MD simulations
3: Materials
4: Methods
5: Notes
6: Utility of MD simulation: A case study on conformational dynamics of d-amino acid oxidase (DAAO)
7: Conclusion and future perspectives
Chapter 27: Applications of molecular dynamics simulations in drug discovery
Abstract
1: Introduction
2: Identification of protein conformation ensemble and drug binding site
3: Modeling protein-drug interactions
4: Modeling drug-membrane interactions
5: Conclusion and future perspectives
Chapter 28: Envisaging the conformational space of proteins by coupling machine learning and molecular dynamics
Abstract
Declaration of competing interests
1: Introduction
2: Conformational impact due to various environment
3: Multiple conformational states of proteins
4: Impact of Ramachandran plot in conformational space
5: Variability in the conformation of intrinsically disordered proteins
6: Conformational sampling analysis through different methods
7: Role of force fields in different conformational space observation
8: Conformational space assessment on the explicit and implicit solvent model
9: Conformational space analysis through machine learning
10: Combination of MD simulation and machine learning
11: Conclusion and future perspectives
Chapter 29: Immunoinformatics and reverse vaccinology methods to design peptide-based vaccines
Abstract
1: Introduction
2: Peptide vaccines
3: Methods and tools in reverse vaccinology
4: Steps involved in reverse vaccinology
5: Advantage of peptide vaccine or multi-epitope vaccines
6: Conclusion and future perspectives
Chapter 30: Computational methods to study intrinsically disordered proteins
Abstract
Acknowledgments
1: Introduction
2: Bioinformatics over biophysical techniques to study IDP
3: Common predictors for identification of IDPs
4: Identification of molecular recognition features (MoRFs)
5: Prediction of nucleic acid-binding regions
6: Biological relevance of predictions
7: Conclusion and future perspectives
Chapter 31: Experimental methods to study intrinsically disordered proteins
Abstract
1: Introduction
2: Size exclusion chromatography
3: UV-vis absorption spectroscopy
4: Circular dichroism spectroscopy
5: Fluorescence spectroscopy
6: Nuclear magnetic resonance spectroscopy
7: Fourier transform infrared spectroscopy
8: Electron spin resonance spectroscopy
9: Raman spectroscopy
10: Light scattering methods
11: Microscopy-based methods
12: Analytical ultracentrifugation
13: Mass spectrometry
14: Conclusions and future perspectives
Chapter 32: Analysis of structure and dynamics of intrinsically disordered regions in proteins using solution NMR methods
Abstract
1: Introduction
2: NMR chemical shift assignments of intrinsically disordered sequences
3: Structural characterization of IDRPs
4: Characterization of IDRP dynamics
5: In-cell NMR experiments
6: Conclusions and future perspectives
Chapter 33: Methods to study the effect of solution variables on the conformational dynamics of intrinsically disordered proteins
Abstract
1: Introduction
2: Computational tools to study the impacts of solution variables on IDPs
Chapter 34: Molecular simulations to study IDP-IDP interactions and their complexes
Abstract
1: Intrinsically disordered proteins and their interactions
2: Introduction of the molecular simulation techniques
3: Characterizing IDP–IDP interactions and their complexes by coarse-grained models
4: Challenges in coarse-grained models
5: Conclusion and future perspectives
Chapter 35: Exploring large-scale protein function using systematic mutant analysis
Abstract
Acknowledgments
1: Introduction
2: Engineering systematic site saturation mutant libraries
3: Screening the systematic mutant libraries for variant function
4: Next-generation sequencing of the variants
5: Large-scale functional mapping in proteins
6: Conclusion and future perspectives
Chapter 36: Approaches and methods to study cell signaling: Linguistics of cellular communication
Abstract
1: Introduction
2: Molecular players in signal transduction
3: Cell signaling can occur in a variety of ways
4: Cell signaling orchestrates key biological processes
5: Experimental techniques used to study cell signaling
6: Conclusion and future perspectives
Chapter 37: Methods to study systems biology of signaling networks: A case study of NSCLC
Abstract
Acknowledgments
1: Introduction
2: Non-small cell lung carcinoma (NSCLC)
3: Systems biology
4: Applications of systems biology approaches in cancer studies
5: System biology methods and approaches
6: Analysis of the data
7: Interpretations and conclusions
Chapter 38: Advancements in the analysis of protein post-translational modifications
Abstract
1: Introduction
2: Ubiquitination
3: Types of ubiquitination
4: Detection of protein ubiquitination
5: Identification of the site of ubiquitination
6: Ubiquitin chain architecture detection
7: Conclusion and future perspectives
Chapter 39: Protein engineering: Methods and applications
Abstract
1: Introduction
2: Protein engineering approaches
3: Directed evolution
4: Semi-rational design
5: De novo design
6: Rational design
7: Applications of protein engineering
8: Conclusions
Chapter 40: Designer 3D-DNA nanodevices: Structures, functions, and cellular applications
Abstract
1: Introduction
2: Different approaches to realize 3D DNA polyhedral nanodevices
3: Methods for characterization of DNA nanostructures
4: Cellular uptake of TDN and its characterization
5: Conclusions and future perspectives
Index

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Timir Tripathi

Vikash Kumar Dubey