Biomolecular and Bioanalytical Techniques

Name: Biomolecular and Bioanalytical Techniques | Theory, Methodology and Applications
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Theory, Methodology and Applications

Vasudevan Ramesh(Herausgeber*in)

Wiley (Verlag)

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Erschienen am 8. März 2019

576 Seiten

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Inhalt

Cover
Title Page
Copyright
Contents
List of Contributors
Preface
Chapter 1 Principles of Health and Safety and Good Laboratory Practice
1.1 Introduction
1.2 Good Laboratory Practice
1.3 Risk Assessment
1.4 Chemical Risk Assessment
1.4.1 Step 1: Identify the Hazards
1.4.2 Step 2: Identify Who Could Be Harmed and How
1.4.3 Step 3: Decide What Controls Are Needed and Whether More Could Be Done
1.4.4 Step 4: Record and Implement the Significant Findings
1.4.5 Step 5: Decide When an Assessment Needs to Be Reviewed
1.5 Biological Materials and Genetically Modified Organisms
1.5.1 Biological Hazards
1.5.2 Genetically Modified Organisms (GMOs)
1.6 Vacuum Apparatus, Pressure Systems and Associated Glassware
1.7 Cryogenic Liquefied Gases
1.8 Compressed Gas Cylinders
1.9 Electromagnetic Radiation
1.9.1 Low Energy or Non-ionising Radiation That Does Not Cause Ionisation of Atoms and Molecules
1.9.2 Ionising Radiation
1.10 Lasers
1.11 High Magnetic Fields
1.12 Sharps
1.13 Ergonomic Issues
1.13.1 Display Screen Equipment
1.13.2 Laboratory Ergonomics
References
Chapter 2 Applications of Chemoinformatics in Drug Discovery
2.1 Significance and Background
2.2 Computer Representation of Chemical Structures
2.3 Database Searching
2.4 Practical Issues on Representation
2.5 Virtual Screening
2.6 Ligand-Based Virtual Screening
2.6.1 Similarity Searching
2.6.2 Scaffold Hopping
2.6.3 3D Similarity Search
2.6.4 Pharmacophore-Based Virtual Screening
2.6.5 Structure-Activity Relationship Modelling
2.7 Protein-Ligand Docking
2.8 Evaluating Virtual Screening Methods
2.9 Case Studies of Virtual Screening
2.9.1 Ligand-Based Virtual Screening: Discovery of LXR Agonists
2.9.2 Combined Ligand- and Structure-Based Approaches: Pim-1 Kinase Inhibitors
2.10 Conclusions
References
Further Reading
Chapter 3 Bioinformatics and Its Applications in Genomics
3.1 Significance and Short Background
3.1.1 Big Data
3.1.2 Computational Challenges
3.1.3 Bioinformatics Roles
3.2 Theory/Principles
3.2.1 Molecular Biology
3.2.2 Gene Structure
3.2.3 Software Development Note
3.3 Databases
3.3.1 Accessing and Using Data
3.4 Techniques
3.4.1 Genome Browsers
3.4.2 Sequence Comparison
3.4.2.1 Similarity and Homology
3.4.2.2 The Dotplot
3.4.2.3 Local and Global Alignment
3.4.2.4 Multiple Sequence Alignment
3.4.2.5 Short Read Aligners - Variant Call Format (VCF)
3.4.2.6 Genome Aligners - BWA
3.4.2.7 Long Read Technologies
3.5 Applications
3.5.1 CRISPR/Cas9 and Off-Targets
3.5.2 Drug Discovery and Target Discovery
3.5.3 Transcriptomics
3.6 Concluding Remarks
References
Further Reading
Websites
Chapter 4 Gene Cloning for the Analysis of Gene Expression
4.1 Identifying Target Sequence
4.2 In Silico Design
4.3 Primer Design
4.3.1 Utility Sequences
4.3.2 Annealing Temperature
4.3.3 Specificity
4.3.4 Additional PCR Primer Design Considerations
4.4 Template Preparation
4.4.1 Genomic DNA Preparation
4.4.2 RNA to Complementary DNA (cDNA) Conversion
4.4.3 Plasmid DNA Preparation
4.4.4 RNA Preparation
4.5 Cloning Methods
4.5.1 Gateway Cloning
4.5.2 Gibson Cloning
4.5.3 Restriction Enzyme Digestion and Ligation Cloning
4.5.4 Ligation Independent Cloning
4.5.5 TA Cloning
4.6 Uses for Cloned DNA Sequences
4.7 Verifying Cloned Sequences
4.7.1 Bacterial Transformation
4.7.2 Mini/Midi/Maxi DNA Prep
4.7.3 Sequence Confirmation
4.8 Applications of Gene Constructs
4.8.1 Recombinant Expression
4.8.1.1 Bacterial System
4.8.1.2 Yeast System
4.8.1.3 Human Cell Transfection
4.8.2 In Vitro Production of RNA
4.9 Case Study: Cloning of a Human Missense Variant Exon into a Minigene Splicing Vector
4.9.1 Methods
4.9.1.1 Prerequisites
4.9.1.2 Background
4.9.1.3 Identification and Amplification of Target Exon
4.9.1.4 In Silico Design
4.9.1.5 Fragment Amplification
4.9.1.6 Bacterial Transformation
4.9.1.7 Plasmid Propagation/Purification
4.9.1.8 HEK Cell Transfection
4.9.1.9 RNA Extraction
4.9.1.10 cDNA Conversion
4.9.1.11 PCR Amplification of Spliced Products
4.10 Case Study: Epitope Tagging of a Yeast Gene
4.10.1 Methods
4.10.1.1 Yeast Genomic DNA Preparation
4.10.1.2 Amplification of Target Yeast Gene
4.10.1.3 Restriction Digestion of PCR Product
4.10.1.4 Restriction Digestion of Plasmid Vector
4.10.1.5 Ligation of DIB1 PCR Product into the pSF-TEF1-COOH-3C-FLAG Plasmid
4.10.1.6 Transformation of Ligation Reaction
4.10.1.7 Plasmid Propagation/Purification
4.10.1.8 Test Restriction Digest and Sequencing
4.10.1.9 Yeast Transformation
References
Further Reading
Websites
Chapter 5 Proteomic Techniques and Their Applications
5.1 Significance and Background
5.2 Principles of Major Proteomics Techniques
5.2.1 MS-Based Techniques
5.2.1.1 Electrospray Ionisation (ESI)
5.2.1.2 Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF)
5.2.2 Proteome Microarray
5.2.3 Bioinformatics Tools and Resources
5.3 Methods for Proteomics
5.3.1 Sample Preparation
5.3.1.1 Cell Sample Preparation
5.3.1.2 Tissue Sample Preparation
5.3.1.3 Blood Sample Preparation
5.3.2 Quantitative Methods
5.3.2.1 Label-Free Quantitative Method
5.3.2.2 Dimethyl Labelling Method
5.3.2.3 iTRAQ Method
5.3.2.4 SILAC Method
5.3.3 Mass Spectrum Data Analysis
5.3.3.1 MaxQuant
5.3.3.2 Mascot
5.3.3.3 Proteome Discoverer
5.4 Applications
5.4.1 Case Study 1: Multiple Proteomics for Discovery of ATP Synthase as a Drug Target
5.4.1.1 ATP Synthase ß-subunit is Upregulated in Tumour Tissues of Breast and Lung Cancer Patients
5.4.1.2 Importance of ATP Synthase
5.4.1.3 2DE-Based Proteomics Reveals Ectopic ATP Synthase Inhibitor Induced Unfolded Protein Response (UPR)
5.4.1.4 Dynamic Phosphoproteomics Identified a Key Phosphosite Involved in Citreoviridin-Induced Pathways
5.4.2 Case Study 2: iTRAQ Quantitative Proteomics to Elucidate Molecular Mechanisms
5.4.2.1 Citreoviridin-Induced Molecular Mechanism in Xenograft Mice Tissue
5.4.2.2 Middle Infrared Radiation-Interfered Networks
5.5 Concluding Remarks
Acknowledgements
References
Further Reading
Website Resources
Chapter 6 Overproduction, Separation and Purification of Affinity-Tagged Proteins from Escherichia coli
6.1 Introduction
6.2 Selecting an Affinity Tag: Glutathione-S-Transferase, Maltose-Binding Protein and Hexa-Histidine Motifs
6.3 The pET Vector Series: Archetypal Expression Vectors in E. coli
6.4 IMAC of a His6-Tagged Protein: Example Methodology with the ParF DNA Segregation Protein
6.4.1 Testing for ParF Induction and Solubility
6.4.2 Identifying the Correct Imidazole Concentration for ParF Protein Elution
6.4.3 Optimised Protocol for ParF Purification
6.4.4 Demonstrating the Functionality of Purified ParF-His6 Protein
6.5 Production and Purification of a GST-Tagged Protein: Example Methodology with the C-Terminal Domain of Yeast RNA Polymerase II
6.5.1 Demonstrating the Functionality of Purified GST-CTD Protein
6.6 Further Purification of Tagged Proteins
6.7 Alternative Hosts for Protein Production
6.8 Concluding Remarks
Acknowledgements
References
Further Reading
Chapter 7 Chromatography: Separation Techniques in Biology
7.1 Introduction to Chromatographic Separation
7.1.1 The Theory of Chromatographic Separation
7.1.2 Chromatography for Biological Samples
7.1.3 Biological Sample Preparation for Chromatography
7.1.4 Extraction and Cleanup Methods for Chromatographic Sample Preparation
7.1.5 Protein Separation
7.2 General Considerations for Protein Separation by Chromatography
7.2.1 Size/Shape - Exploited by Size Exclusion Chromatography (SEC)
7.2.2 Charge - Exploited by Ion Exchange Chromatography (IEX)
7.2.3 Hydrophobicity - Exploited by Hydrophobic Interaction Chromatography (HIC) and Reversed-Phase Chromatography (RPC)
7.2.4 Biological Function and/or Structure - Exploited by Affinity Chromatography
7.3 Engineering Proteins for Streamlined Chromatographic Separations
7.4 Example Chromatographic Separations of Biological Samples
7.4.1 On-Column Protein Folding
7.4.2 On-Column Protein Refolding via Anion Exchange Chromatography
7.4.3 Protein Structure Elucidation
7.5 Other Applications of Chromatography for Biological Sample Preparation and Analysis
References
Further Reading
Chapter 8 Synthetic Methodology in Chemical Biology
8.1 Introduction
8.2 Peptide Synthesis
8.3 Amide Bond Synthesis
8.3.1 Solid Phase Peptide Synthesis (SPPS)
8.3.2 Native Chemical Ligation (NCL)
8.3.3 Reactions of Endogenous Amino Acids
8.3.4 Dehydroalanine (Dha) Procedure
8.4 Bioorthogonal Chemistry
8.4.1 The Staudinger Ligation
8.5 The Copper-Catalysed Azide-Alkyne Cycloaddition Reaction (CuAAC)
8.5.1 The Strain-Promoted Azide-Alkyne Cycloaddition Reaction (SPAAC)
8.5.2 Tetrazine Ligations
8.6 Unnatural Amino Acid Incorporation
8.7 Case Studies
8.7.1 Post-Translational Modification of Histone Tails
8.7.1.1 Cysteine Alkylation
8.7.1.2 Native Chemical Ligation (NCL)
8.7.1.3 Genetically Encoded Methyl-Lys Incorporation
8.8 Conclusion
8.8.1 Overall Conclusion
References
Further Reading
Chapter 9 Reaction Chemical Kinetics in Biology
9.1 Significance
9.1.1 Examples of Enzyme Use (Clinical and Biotechnology)
9.2 Overview of Kinetics and Its Application to Biology
9.2.1 Steady-State Kinetics
9.2.2 Beyond the Michaelis-Menten Equation
9.2.2.1 Enzyme Cooperativity
9.2.2.2 Multiple Substrates
9.3 Determination of Enzyme Kinetic Mechanisms
9.3.1 Michaelis-Menten Parameters
9.3.2 Cooperative Enzymes
9.3.3 Substrate Inhibition
9.3.4 Determination of the Mechanism of a Bisubstrate Enzyme
9.4 Technique/Protocol: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme and Use of Product Inhibition to Determine Mechanism
9.4.1 Method Requirements
9.4.2 Procedure
9.4.2.1 Determine Suitable Concentrations of the Enzyme to be Used
9.4.2.2 Determine Estimates of KM for both Substrates
9.4.2.3 Determine the Kinetic Parameters for both Substrates Accurately
9.4.2.4 Determining the Mechanism of the Bisubstrate Enzyme
9.4.2.5 Determining which Sequential Mechanism Is Adopted by a Bisubstrate Enzyme
9.5 Case Study: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme
9.6 More Advanced Methods
9.7 Concluding Remarks
References
Chapter 10 Mass Spectrometry and Its Applications
10.1 Significance
10.2 Theories and Principles of Biomolecular Mass Spectrometry
10.2.1 Gas-Phase Ion Generation of Biomolecules
10.2.1.1 Electron Ionisation
10.2.1.2 Inductively Coupled Plasma
10.2.1.3 Electrospray Ionisation
10.2.1.4 Matrix-Assisted Laser Desorption Ionisation
10.2.2 Mass Analysers
10.2.2.1 Sector Analysers
10.2.2.2 Time-of-Flight Analysers
10.2.2.3 Quadrupole Mass Filters
10.2.2.4 Quadrupole Ion Traps
10.2.2.5 Ion Cyclotron Resonance
10.2.2.6 Orbitrap
10.2.3 Ion Detection
10.2.4 Hybrid Instrumentation
10.3 Techniques and Methodology in Biomolecular Mass Spectrometry
10.3.1 Tandem Mass Spectrometry
10.3.2 Gas-Phase Ion Activation
10.3.2.1 Collision-Induced Dissociation
10.3.2.2 Other Activation Methods
10.3.3 Solution Phase Chemistry
10.3.3.1 Covalent Labelling
10.3.3.2 Chemical Cross-Linking
10.3.3.3 Hydrogen-Deuterium Exchange
10.3.4 Experimental Considerations for Biomolecular Mass Spectrometry
10.4 Applications
10.4.1 Elemental Analysis
10.4.2 Mass Spectrometry of Organic Compounds
10.4.3 Mass Spectrometry for Macromolecules
10.5 Concluding Remarks
References
Further Reading
Chapter 11 Applications and Complementarity of Analytical Ultracentrifugation and Light-Scattering Techniques
11.1 Introduction
11.2 Analytical Ultracentrifugation
11.2.1 Information Content of an AUC Experiment
11.2.2 The AUC Experiment
11.2.3 AUC Theory and Data Analysis
11.3 Light Scattering
11.3.1 Information in LS Experiments
11.3.2 The LS Experiment
11.3.3 LS Theory and Data Analysis
11.3.3.1 Static Light Scattering
11.3.3.2 Dynamic Light Scattering
11.4 Protocols
11.4.1 DLS/SLS Protocol
11.4.2 SEC-MALS Protocol
11.4.3 SV Protocol
11.5 Applications
11.5.1 Tp0037
11.5.1.1 DLS on Tp0037
11.5.1.2 SLS on Tp0037
11.5.1.3 SV on Tp0037
11.5.2 BSA
11.5.2.1 DLS on BSA
11.5.2.2 SEC-MALS on BSA
11.5.2.3 SV on BSA
11.5.3 Complementarity Between AUC and LS
11.5.4 Interacting Systems in LS and AUC
11.6 Conclusions
Acknowledgements
References
Further Reading
Chapter 12 Application of Isothermal Titration Calorimetry (ITC) to Biomolecular Interactions
12.1 Introduction
12.1.1 Why Measure Binding of Biological Molecules?
12.1.2 Approaches for Analysis of Biomolecular Interactions
12.1.3 Why Use ITC?
12.2 Principles and Theory of ITC
12.2.1 What Is ITC and What Does It Measure?
12.2.2 Overview of Current ITC Microcalorimeters
12.2.3 ITC and Binding Theory
12.3 Protocols for Design, Implementation and Analysis of ITC Experiments
12.3.1 Where to Begin? Planning a First ITC Experiment
12.3.2 Sample Requirements and Preparing for an ITC Experiment
12.3.2.1 Sample Preparation
12.3.2.2 Buffer Considerations and Matching of Solutions
12.3.2.3 Importance of Determining Accurate Sample Concentrations
12.3.3 Running the ITC Experiment
12.3.3.1 Sample Loading and Experiment Set Up on a VP-ITC
12.3.3.2 Sample Loading and Experiment Set Up on an Auto-iTC200
12.3.3.3 Dealing with Ligand 'Heats of Dilution'
12.3.3.4 Troubleshooting Some Common Issues
12.3.4 ITC Data Analysis and Fitting to a Binding Model
12.4 Example Applications of ITC to Analysis of Biomolecular Interactions
12.4.1 Case Study 1: Enthalpy versus Entropy Driven Transcription Factor (TF) Binding to Distinct DNA Sequences
12.4.2 Case Study 2: Identification of a New Motif Essential for Co-substrate S-adenosyl-L-methionine (SAM) Binding by an rRNA Methyltransferase
12.5 Concluding Remarks
Acknowledgements
References
Further Reading
Website Resources
Chapter 13 An Introduction to Infra-red and Raman Spectroscopies for Pharmaceutical and Biomedical Studies
13.1 Significance and Short Background
13.2 Theory
13.2.1 Electromagnetic Radiation
13.2.2 Molecular Vibration
13.2.3 Vibrational Energy
13.2.4 Modes of Vibration
13.2.5 Number of Vibrations
13.2.6 Infra-red Spectroscopy
13.2.6.1 Infra-red Spectrum
13.2.6.2 Transmittance versus Absorbance
13.2.7 Raman Spectroscopy
13.3 Technique/Methodology/Protocol
13.3.1 Infra-red Spectrometer
13.3.2 Infra-red Sampling Protocol
13.3.2.1 Reflection Measurements
13.3.2.2 Transmission Measurements
13.3.2.3 Attenuated Total Reflection (ATR)
13.3.2.4 Multireflection ATR
13.3.2.5 FTIR Microscope
13.3.3 Raman Scattering
13.3.3.1 Spontaneous Raman Scattering
13.3.3.2 Resonance Raman Scattering
13.3.3.3 Non-linear Raman Scattering
13.3.3.4 Surface Enhanced Raman Scattering
13.3.4 Raman Spectrometer
13.3.5 Raman Sampling Protocol
13.4 Applications
13.4.1 Case Study 1: Drug Polymorph Studies
13.4.1.1 Amorphous Drug Studies
13.4.2 Case Study 2: Live Cell Analysis
13.4.2.1 Live Cell Studies by Raman Spectroscopy
13.4.2.2 Live Cell Study by FTIR Spectroscopy
13.5 Concluding Remarks
References
Further Reading
Chapter 14 Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes
14.1 Significance and Background
14.2 Theory and Principles
14.2.1 Electromagnetic Radiation and Molecular Energy Levels
14.2.2 The Fluorescence Sequence
14.2.2.1 Step 1: Light Absorption
14.2.2.2 Step 2: Relaxation to the Equilibrium Excited State
14.2.2.3 Step 3: De-excitation of the Excited State and Fluorescent Photon Emission
14.2.2.4 Step 4: Vibrational Relaxation to the Ground State
14.2.3 Information Content of Fluorescence Excitation/Emission Spectra
14.2.4 Solvent Effects
14.3 Techniques, Methodologies and Protocols
14.3.1 Instrumentation
14.3.2 Fluorophores
14.3.3 Measuring Fluorescence Spectra
14.3.4 Fluorescence Quenching
14.3.4.1 Fluorescence Quenching Sample Experiment: Iodide Quenching of Tryptophan Fluorescence
14.3.4.2 Experimental Setup and Data Collection
14.3.4.3 Data Analysis
14.3.4.4 Experimental Considerations
14.3.5 Förster Resonance Energy Transfer (FRET)
14.3.5.1 FRET Sample Experiment: Monitoring Molecular Interactions
14.3.5.2 Experimental Setup and Data Collection
14.3.5.3 Data Analysis
14.3.5.4 Experimental Considerations
14.3.6 Fluorescence Anisotropy
14.3.6.1 Anisotropy Sample Experiment: Analysis of Molecular Rotational Dynamics
14.3.6.2 Experimental Setup and Data Collection
14.3.6.3 Data Analysis
14.3.6.4 Experimental Considerations
14.4 Case Studies: Fluorescence Spectroscopy to Analyse Membrane Protein Structural Dynamics
14.4.1 Site-specific Labelling of Tim23 by Cotranslational Probe Incorporation
14.4.2 Fluorescence Analysis of Tim23 Structural Features
14.4.3 Fluorescence Analysis of Tim23 Conformational Dynamics
14.5 Concluding Remarks
Acknowledgements
References
Further Reading
Chapter 15 Circular Dichroism and Related Spectroscopic Techniques
15.1 Significance and Background
15.2 Theory/Principles
15.2.1 Circular Dichroism Spectroscopy
15.2.2 Linear Dichroism
15.3 Technique/Methodology/Protocol
15.3.1 The Sample: Concentration, Pathlength and Matrix
15.3.2 The Instrument
15.3.2.1 UV/Visible Instruments
15.3.2.2 Infra-red Absorbance
15.3.2.3 Linear Dichroism
15.3.3 Structure Fitting of Proteins from Spectral Data
15.4 Applications
15.4.1 Comparison of Different a-Lactalbumin Protein Structures
15.4.2 Linear Dichroism Examples
15.4.2.1 Flow Oriented DNA and Ligand Binding
15.4.2.2 M-13 Bacteriophage
15.5 Concluding Remarks
References
Further Reading
Chapter 16 Principles and Practice in Macromolecular X-Ray Crystallography
16.1 Significance and Short Background
16.2 Theory and Principles: Overview
16.2.1 Basics of Crystallisation
16.2.1.1 Crystallisation
16.2.2 Basics of X-Ray Crystallography
16.2.2.1 Bragg's Law
16.2.3 Scaling
16.2.4 Radiation Damage
16.2.5 Matthews' Coefficient
16.2.6 Non-crystallographic Symmetry
16.2.7 Structure Factors
16.2.8 Phase Problem
16.2.8.1 Molecular Replacement
16.2.8.2 Experimental Phasing: SAD
16.2.8.3 Experimental Phasing: MAD
16.2.8.4 Experimental Phasing: SIR and MIR
16.2.9 Model Building and Refinement
16.3 Methodology
16.3.1 Construct Design
16.3.2 Crystallisation Screening
16.3.2.1 Crystallisation Optimisation
16.3.2.2 Crystallisation Seeding
16.3.3 Cryoprotection
16.3.3.1 Mounting
16.3.3.2 Cryocooling
16.3.3.3 Storage and Transportation
16.3.4 Data Collection Strategies
16.3.4.1 Mounting and Centring the Sample
16.3.4.2 Collecting the First Few Images and Indexing
16.3.4.3 Sacrificial Sample
16.3.4.4 Indexing and Integration
16.3.4.5 Scaling
16.3.5 Native Data Collection, Molecular Replacement and Ligand Soaks
16.3.5.1 Experimental Phasing
16.3.5.2 Single Wavelength Anomalous Scattering - SAD
16.3.5.3 Multiple Wavelength Anomalous Scattering - MAD
16.3.5.4 Other Experimental Phasing Techniques
16.3.6 Data Processing
16.3.6.1 Difference Fouriers
16.3.6.2 Molecular Replacement
16.3.6.3 Experimental Phasing
16.3.7 Model Building and Refinement
16.3.7.1 Validation
16.3.7.2 Deposition and Publication
16.4 Applications
16.4.1 Molecular Replacement, the Latest Automated Software Approaches and Drug Discovery
16.4.2 Use of Sulphur as an Anomalous Scatterer for Solving the Phase Problem
16.5 Concluding Remarks
Acknowledgements
References
Further Reading
Chapter 17 Biomolecular NMR Spectroscopy and Structure Determination of DNA
17.1 Significance and Background
17.1.1 Analytical NMR: Discovery and Evolution
17.1.2 The Aim of the Chapter
17.2 Basic NMR Theory
17.2.1 Basic Principles of NMR
17.2.2 NMR Experimental Parameters
17.2.2.1 Chemical Shift
17.2.2.2 Line Integral
17.2.2.3 Spin-Spin Coupling
17.2.3 Longitudinal (T1) and Transverse Relaxation (T2)
17.2.4 Nuclear Overhauser Effect (NOE)
17.2.5 NMR Rate Processes
17.3 Multidimensional NMR Spectroscopy
17.3.1 One-Dimensional NMR (1D NMR)
17.3.2 Two-Dimensional NMR (2D NMR)
17.3.3 Three-Dimensional NMR (3D NMR)
17.4 NMR Instrumentation and Experiments
17.4.1 NMR Instrumentation
17.4.2 NMR Experiments
17.4.2.1 Solvent Water Suppression by Pre-saturation
17.4.2.2 Solvent Water Suppression by Gradient Method
17.4.2.3 X 1H Decoupled 1D Experiment
17.4.2.4 Correlated Spectroscopy (COSY)
17.4.2.5 Double Quantum Filtered Correlated Spectroscopy (DQF-COSY)
17.4.2.6 Total Correlation Spectroscopy (TOCSY)
17.4.2.7 Nuclear Overhauser Effect Spectroscopy (NOESY)
17.4.2.8 Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)
17.4.2.9 CPMG-HSQC-NOESY
17.5 Structure and Conformational Parameters of DNA
17.5.1 Basic Building Blocks of DNA
17.5.2 Phosphodiester Linkage
17.5.3 Watson-Crick Hydrogen Bonded Base Pairs
17.5.4 Models of DNA
17.5.4.1 B-DNA
17.5.4.2 A-DNA
17.5.4.3 Z-DNA
17.5.5 Conformation of DNA
17.6 NMR Structure Determination [50-55]
17.6.1 NMR Assignment Strategy for DNA
17.6.2 Molecular Modelling
17.6.3 Distance Constraints
17.6.4 Dihedral Angle Constraints
17.6.5 Hydrogen Bond/Planarity Constraints
17.6.6 Structure Calculations
17.6.7 Conformational Analysis
17.6.8 Deposition of Structures in Databases
17.7 Case Study 1: NMR Structure Determination and Conformational Analysis of 17mer Canonical GC DNA
17.7.1 Sample Preparation of 17mer GC DNA
17.7.2 Acquisition, Processing and Analysis of 17mer GC DNA NMR
17.7.2.1 Data Acquisition
17.7.2.2 NMR Data Processing and Analysis
17.7.3 NMR Assignment of 17mer-GC DNA
17.7.3.1 Identification and Assignment of Exchangeable Proton Resonances
17.7.3.2 Identification and Assignment of Non-exchangeable Proton and Carbon Resonances
17.7.4 Table of Assignments
17.7.5 Determination of Experimental Constraints for Structure Calculation
17.7.6 Structure Calculation
17.7.7 Conformational Analysis
17.8 Case Study 2: NMR Structure Determination and Conformational Analysis of 13mer 6-Thioguanine Modified GC DNA [70-74]
17.8.1 Structure of 6-Thioguanine DNA
17.8.2 NMR Assignment of 13mer 6-Thioguanine Modified GC DNA
17.8.2.1 Identification and Assignment of Exchangeable Proton Resonances
17.8.3 Table of Assignments
17.8.4 Structure Calculation
17.8.5 Conformational Analysis
17.9 Conclusion
References
Further Reading
Chapter 18 Cryo-TEM and Biological Structure Determination
18.1 Significance and Background
18.2 Theoretical Principles of Biological Cryo-TEM
18.2.1 Use of Electron Radiation for Imaging of Biological Machinery
18.2.2 Electron Beam Interaction with a Biological Sample
18.2.3 Image Formation: The Weak Phase Object Challenge
18.2.4 Basic Anatomy of a TEM
18.2.5 Systematic TEM Defects and Their Influence on Image Formation
18.2.6 CTF Detection and Correction
18.2.7 Resolution Limiting Factors
18.3 Experimental Approaches in Biological Cryo-TEM
18.3.1 Sample Preparation for Cryo-TEM
18.3.1.1 Sample Preparation of Thicker Specimens
18.3.2 Cryo-TEM Data Collection
18.3.2.1 Image Acquisition for Cryo-TEM: The Resolution Revolution with Direct Electron Detectors (DEDs)
18.3.2.2 Data Collection Strategies for SPA
18.3.2.3 Data Collection Strategies for Cryo-Electron Tomography (Cryo-ET)
18.3.2.4 Special Cases of Cryo-TEM Samples and Data Collection Experiments
18.3.3 Image Processing for Cryo-TEM Samples
18.3.3.1 Cryo-TEM Pre-processing
18.3.3.2 Structure Determination Strategies for SPA
18.3.3.3 Structure Determination Strategies for Cryo-ET and Use of Subtomogram Averaging
18.3.3.4 Resolution Determination and Structure Interpretation
18.4 Cryo-TEM Case Studies
18.4.1 Case Study 1: Single Particle Analysis of Microtubule Stabilisation by a Neuronal Microtubule-Associated Protein Doublecortin
18.4.1.1 DCX-MT Sample Preparation and Cryo-TEM Data Collection
18.4.1.2 Image Processing and 3D Reconstruction
18.4.2 Case Study 2: Insight into the Human Immunodeficiency Virus Type 1 (HIV-1) Assembly Using Subtomogram Averaging
18.4.2.1 Virus Sample Preparation and cryo-ET Data Collection
18.4.2.2 Resolution Improvement Factors
18.5 Concluding Remarks
Acknowledgements
References
Website Resources
Chapter 19 Computer Modelling and Molecular Dynamics Simulation of Biomolecules
19.1 Significance
19.2 Theory and Principles
19.2.1 General Considerations
19.2.2 Statistical Mechanics: Partition Function and Ensemble Average
19.2.3 Classical Mechanics: Equations of Motion
19.3 Methodology
19.3.1 Molecular Mechanics: Biomolecular Force Fields
19.3.2 Molecular Dynamics Simulation of Biomolecules
19.3.2.1 Common Algorithms
19.3.2.2 Special Topics
19.4 Applications
19.4.1 Comparative Protein Structure Modelling
19.4.2 Alchemical Free-Energy Simulations to Identify Key Residues of Protein-Protein Interaction
19.5 Concluding Remarks
References
Further Reading
Index
EULA

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