Essentials of Chemical Biology

Links 2.4.3 Solid Phase Oligosaccharide Synthesis 2.5 Chemical Synthesis of Lipids 2.6 Biological Synthesis of Biological Macromolecules 2.6.1 Ion Exchange Chromatography 2.6.2 Hydrophobic Interaction Chromatography 2.6.3 Reversed-Phase Chromatography 2.6.4 Gel Filtration Chromatography 2.6.5 Hydroxyapatite Chromatography 2.7 Directed Biological Synthesis of Proteins 2.7.1 Wild-type or Recombinant Sources 2.7.2 Expression in E. coli; Early Purification 2.7.3 Affinity Chromatography 2.7.3.1 Immobilised Metal Affinity Chromatography 2.7.3.2 Glutathione-S-Transferase Tags 2.7.3.3 Maltose Binding Protein Tags 2.7.3.4 Biotinylation of Proteins 2.7.3.5 Intein Tags 2.7.3.6 Other Affinity Tags and Radiolabelling of Proteins 2.8 Biological Syntheses of Nucleic Acids, Oligosaccharides and Lipids 2.8.1 Biological Synthesis of Nucleic Acids 2.8.2 Biological Synthesis of Oligosaccharides 2.8.3 Biological Synthesis of Lipids Chapter 3 Molecular Biology as a Toolset for Chemical Biology 3.1 Key Concepts in Molecular Biology 3.1.1 The Central Dogma of Molecular Biology 3.1.2 The Difference between Prokaryotic and Eukaryotic Genes 3.1.3 The Creation of cDNA Libraries 3.2 Tools and Techniques in Molecular Biology 3.2.1 Plasmid DNA Vectors 3.2.2 Restriction Enzymes 3.2.3 DNA Ligases 3.2.4 Hosts 3.2.5 Cellular Transformation 3.2.6 Selection 3.2.7 pDNA Purification 3.2.8 Nucleic Acid Electrophoresis 3.2.9 DNA Sequencing 3.3 Cloning and Identification of Genes in DNA 3.3.1 Direct DNA Cloning 3.3.2 Polymerase Chain Reaction 3.3.3 Gene Expression and Expression Vectors 3.3.3.1 Expression Vectors 3.3.3.2 Protein Expression Strategy 3.3.3.3 Cloning for RNA Synthesis 3.4 Integrating Cloning and Expression 3.4.1 Designing Forward and Reverse Primers 3.4.2 PCR Amplification and Product Isolation 3.4.3 Ligation and Transformation 3.4.4 Validation and Sequencing 3.4.5 Protein Expression 3.4.6 Cloning and Expressing from Eukaryotic Genes 3.5 Site Directed Mutagenesis 3.5.1 PCR-based Approaches to Mutagenesis 3.5.2 Non-PCR-based Approaches to Mutagenesis Chapter 4 - Electronic and Vibrational Spectroscopy 4.1 Electronic and Vibrational Spectroscopy in Chemical Biology 4.2 UV-Visible Spectroscopy 4.2.1 Transition Dipole Moments 4.2.2 UV-Visible Spectroscopy of Proteins 4.2.3 UV-Visible Spectroscopy of Nucleic Acids 4.2.4 Structural vs Functional Information from UV-Visible Spectroscopy 4.3 Circular Dichroism Spectroscopy 4.3.1 Circularly Polarised Light 4.3.2 Optical Activity and Circular Dichroism 4.3.3 The Circular Dichroism Spectrum 4.3.4 Structural vs Functional Information from Circular Dichroism Spectroscopy 4.4 Vibrational Spectroscopy 4.4.1 Infra-Red Vibrational Modes 4.4.2 Structural Information from Infra-Red Spectroscopy 4.4.3 Raman Spectroscopy 4.5 Fluorescence Spectroscopy 4.5.1 Rates of Emission and Lifetimes 4.5.2 Effects of Non-Radiative Competition Processes 4.5.3 Structural vs Functional Information from Fluorescence Spectroscopy 4.5.4 Extrinsic Fluorescence and FRET 4.5.5 Probing Biological Macromolecule Functions with Extrinsic Fluorescence 4.5.5.1 Chemical Conjugation of Extrinsic Fluorescent Probes 4.5.5.2 Biological Conjugation of Extrinsic Fluorescent Probes 4.5.5.3 Selecting Extrinsic Fluorescent Probes 4.5.6 Fluorescence Single Molecule Spectroscopy (SMS) 4.6 Probing Metal Centres in Biological Systems by Spectroscopy Chapter 5 - Magnetic Resonance 5.1 Magnetic Resonance in Chemical Biology 5.2 Key Principles of NMR 5.2.1 Spin Angular Momentum 5.2.2 Magnetic Moment 5.2.3 Quantum Mechanical Description of NMR 5.2.4 Chemical Shift and Coupling 5.2.4.1 Chemical Shift 5.2.4.2 Spin-Spin Coupling 5.2.5 Vector Description of NMR 5.2.6 Spin-Lattice and Spin-Spin Relaxation 5.2.7 Nuclear Overhauser Effect 5.3 Two-dimensional NMR 5.3.1 Homonuclear 2-D COSY and TOCSY Experiments 5.3.2 Heteronuclear Correlation Experiments 5.3.3 NOESY Experiments 5.4 Multi-dimensional NMR 5.4.1 Basic Principles of 3D Experiments 5.4.2 Correlation Experiments 5.4.3 Basic Principles of 4D Experiments 5.5 Biological Macromolecule Structural Information 5.5.1 Analysing Protein Structures 5.5.1.1 3-D COSY and TOCSY Experiments of Proteins 5.5.1.2 3-D HNCA Experiment of Proteins 5.5.1.3 3D- and 4D-NOESY Experiments of Proteins 5.5.1.4 Energy Minimisations 5.5.1.5 Techniques for Overcoming the Molecular Weight Limit 5.5.2 Analysing Nucleic Acid Structures 5.5.3 Analysing Carbohydrate Structures 5.5.4 Analysing Lipid Assembly Structures 5.6 EPR Spectroscopy; Key Principles 5.6.1 Quantum Mechanical Description of EPR 5.6.2 g-Value 5.6.3 Hyperfine Splitting 5.6.4 Biological Macromolecule Structural Information Chapter 6 - Diffraction and Microscopy 6.1 Diffraction and Microscopy in Chemical Biology 6.2 Key Principles of X-ray Diffraction 6.2.1 Unit Cell 6.2.2 Bragg Law 6.2.3 Reciprocal Lattice 6.2.4 Structure Factors 6.2.5 The Phase Problem 6.2.6 Harker Construction 6.3 Structural Information from X-ray Diffraction 6.3.1 Biological Macromolecule Crystallisation 6.3.2 X-Ray Generation 6.3.3 Determination of X-ray Diffraction Pattern 6.3.4 Heavy Atom Derivitisation 6.3.5 Fitting an Electron Density Map 6.3.6 Biological Macromolecular Structures by X-ray Crystallography 6.4 Neutron Diffraction 6.5 Key Principles of Electron Microscopy 6.5.1 Duality of Matter 6.5.2 Electron Wavelengths 6.5.3 Sample Preparation 6.5.4 Contrast Imaging 6.5.5 Image Processing 6.5.6 Biological Macromolecular Structures from Electron Microscopy 6.6 Key Principles of Scanning Probe Microscopy 6.6.1 STM Concept 6.6.2 Electron Tunnelling 6.6.3 Piezo Electric Drives 6.6.4 STM Scanning Modes 6.6.5 Origin of AFM 6.6.6 AFM Cantilever 6.6.7 AFM Scanning Modes 6.6.8 Biological Structural Information from STM and AFM Chapter 7 - Molecular Recognition and Binding 7.1 Molecular Recognition and Binding in Chemical Biology 7.1.1 Roles of Molecular Recognition and Binding 7.1.1.1 Acetyl Choline, Receptor and Esterase 7.1.1.2 Adaptive Immunity, Antibodies and Myasthenia Gravis 7.1.1.3 DNA Packaging and Expression Control 7.1.2 Theoretical Framework for Molecular Recognition and Binding 7.1.2.1 Motion in Solution 7.1.2.2 Long Range Molecular Recognition 7.1.2.3 Short Range Molecular Recognition and Binding 7.2 Theoretical Models of Binding 7.2.1 Single Site Single Affinity Binding 7.2.2 Independent Multiple Site, Equal Affinity Binding 7.2.3 Independent Multiple Site, Variable Affinity Binding 7.2.4 Dependent Multiple Site Cooperative Binding and Hill Equation 7.3 Analysing Molecular Recognition and Binding 7.3.1 Equilibrium Dialysis 7.3.2 Titration Methodologies 7.3.2.1 Titration Data Estimates 7.3.2.2 Physical Properties vs Spectroscopic Signatures 7.3.3 Isothermal Titration Calorimetry and Binding Thermodynamics 7.3.3.1 Equilibrium Thermodynamics of Molecular Recognition and Binding 7.3.3.2 Enthalpy of Binding and ITC 7.3.3.3 Van't Hoff Relationships 7.3.4 Capillary Electrophoresis 7.3.5 Resonant Mirror Biosensing (Surface Plasmon Resonance) 7.4 Biological Molecular Recognition Studies 7.4.1 LysU Enzyme Substrate Recognition 7.4.2 Stress Protein Molecular Chaperones 7.4.2.1 GroEL 7.4.2.2 Hsp47 7.4.3 Complementary Peptides Chapter 8 - Kinetics and Catalysis 8.1 Catalysis in Chemical Biology 8.1.1 Simple Principles in Biocatalysis 8.1.2 Steady State Kinetics in Biocatalysis 8.1.3 Steady-state Bioassays 8.2 Steady State Kinetic Schemes 8.2.1 Simple Steady State Kinetics and Michaelis-Menten Equation 8.2.2 Interpretation of kcatand KM 8.2.3 Determination of kcatand KM 8.2.4 Effect of Steady State Inhibitors 8.2.4.1 Competitive Inhibition 8.2.4.2 Non-competitive Inhibition 8.2.4.3 Un-competitive Inhibition 8.2.5 Applicability of Michaelis-Menten Equation 8.2.6 Multiple Substrate/Product Steady State Kinetics 8.2.6.1 Multiple Catalytic Sites, Non-cooperative Uni-Uni Kinetic Scheme 8.2.6.2 Multiple Catalytic Sites, Cooperative Uni Uni Kinetic Scheme and Hill Equation 8.2.6.3 Ordered Uni-Bi Kinetic Scheme 8.2.6.4 Ordered Bi-Uni Kinetic Scheme 8.2.7 Multiple Substrate/Product King-Altman Kinetics 8.2.7.1 Reversible Uni-Uni Kinetic Scheme 8.2.7.2 Ordered Bi-Bi Kinetic Scheme 8.2.7.3 Ping-Pong Bi-Bi Kinetic Scheme 8.2.7.4 Ordered Ter Bi and Ter Ter Kinetic Scheme 8.3 Pre-steady State Kinetics 8.3.1 Pre-steady State Bioassays 8.3.2 First-Order Pre-steady State Equations 8.3.3 Further Pre-steady State Equations 8.4 Theories of Biocatalysis 8.4.1 Intramolecular Catalysis and Stereo-control in Catalysis 8.4.2 "Orbital Steering" 8.4.3 Induced-Fit and Strain 8.4.4 General-Acid-Base Catalysis 8.4.4.1 Dixon-Webb Log Plots 8.4.5 Electrophilic and Nucleophilic Catalysis 8.46 Mechanisms of Biocatalysis by Selected Biocatalysts 8.47 Transition State Stabilisation and Biocatalysis 8.4.7.1 Basic Transition-State Concepts 8.4.7.2 Binding Energy in Biocatalysis 8.4.8 "Perfect Biocatalyst" Theory 8.4.9 Linear-Free Energy Relationships 8.5 Electron Transfer 8.5.1 Electron Transfer Kinetics 8.5.2 Electron Transfer Step Chapter 9 - Mass Spectrometry and Proteomics 9.1 Mass Spectrometry in Chemical Biology 9.2 Key Principles in Mass Spectrometry 9.2.1 Ionisation Sources 9.2.1.1 Traditional Techniques of Ionisation 9.2.1.2 Desorption Ionisation Techniques - FAB and MALDI 9.2.1.3 Spray Ionisation Techniques - Thermospray and Electrospray 9.2.2 Mass Analysers in Mass Spectrometry 9.2.2.1 Time of Flight (TOF) Mass Analysers 9.2.2.2 Quadrupole Mass Analysers 9.2.2.3 Ion Trap Mass Analysers 9.2.2.4 Fourier Transform Ion Cyclotron Resonance (FTICR) and Orbitrap Mass Analysers 9.2.2.5 Tandem Mass Analysers (MS/MS) 9.3 Structural Analysis of Biological Macromolecules and Lipids by Mass Spectrometry 9.3.1 Analysis of Individual Peptides by Mass Spectrometry 9.3.2 Analysis of Proteins by Mass Spectrometry 9.3.2.1 Protein Molecular Weight Determination 9.3.2.2 Gel-Based Isolation and Digestion of a Protein for Mass Spectrometry 9.3.2.3 Peptide Mass Fingerprinting for Protein Identification 9.3.2.4 Tandem Mass Spectrometery for Protein Identification 9.3.3 Analysis of Oligonucleotides by Mass Spectrometry 9.3.4 Analysis of Carbohydrates and Glycoproteins by Mass Spectrometry 9.3.5 Analysis of Lipids by Mass Spectrometry 9.4 The Challenge of Proteomics 9.4.1 Early Developments in Proteomics 9.4.2 Using 2-D Gel Electrophoresis with Mass Spectrometry 9.4.3 Isotope-Coded Affinity Tags 9.4.4 Deciphering Protein Networks by Tandem Affinity Purification 9.4.5 The Challenge of Membrane Proteins in Proteomics 9.4.6 Proteomics and Post-Translational Modifications 9.4.6.1 Comprehensive PTM Analysis of a Single Protein 9.4.6.2 PTM Analysis of Protein Populations 9.5 Genomics - Assigning Function to Genes and Proteins 9.5.1 Protein Microarrays 9.5.1.1 Analytical Arrays 9.5.1.2 Functional Arrays 9.5.2 Biochemical Genomics 9.5.3 Chemical Genomics 9.5.4 Structural Genomics 9.5.5 Perspectives on the Future of Proteomics with Genomics Chapter 10 - Molecular Selection and Evolution 10.1 Chemical Biology and the Origins of Life 10.1.1 Order from Complexity 10.1.2 Evolution from the Molecular Level 10.1.3 Chemical Self-Organisation from Complexity 10.1.4 Origins of Biological Macromolecules of Life 10.2 Molecular Breeding; Natural Selection Acting on Self-Organisation 10.3 Directed Evolution of Protein Function 10.3.1 Random Mutagenesis and PCR 10.3.2 Mutagenesis and DNA Shuffling 10.3.3 Oligodeoxynucleotide Cassette Mutagenesis 10.3.4 Screening Strategies * Directed Evolution of Nucleic Acids 10.4.1 Aptamers 10.4.1.1 Design and Construction of Polydeoxynucleotide/Polynucleotide Libraries 10.4.1.2 Partition, Amplification and Iteration 10.4.1.3 Applications of Aptamers 10.4.2 Selection of Catalytic RNA 10.4.3 DNA Aptamers and Catalytic DNA 10.5 Catalytic Antibodies Chapter 11 - Chemical Biology of Cells 11.1 General Introduction 11.2 Array Technologies, Microfluidics, and Miniaturization 11.2.1 Arrays from Past to Present 11.2.2 Micropatterned and Microfluidic Devices 11.3 Chemical Genetics and Potential New Therapeutics 11.3.1 Early Chemical Genetics 11.3.2 More Recnt Chemical Genetics 11.3.2.1 Chemical Genetics in MoA Identification 11.3.2.2 Chemical Genetics in Drug Resistance and Drug-Drug Interactions 11.4 Chemical Cellular Dynamics 11.5 Chemical Biology and in vivo Cell Connectomics 11.5.1 Brainbow Connectomics 11.5.2 Brainbow Applications Chapter 12 - Chemical Biology of Stem Cells to Tissue Engineering 12.1 General Introduction 12.2 Chemical Stem Cell Biology 12.2.1 Stem Cell Regulation 12.2.1.1 Biochemical Regulation 12.2.1.2 Biophysical Regulation 12.2.2 Controlling Stem Cell Regulation by Biochemical and Biophysical Means 12.2.3 Controlling Stem Cell Regulation by Genetic Means 12.2.4 Stem Cell Modelling 12.2.4.1 Deterministic Modelling 12.2.4.2 Other Modelling Approaches 12.3 The Road to Cell Therapies 12.3.1 The Need for Bioreactors 12.3.2 Practical Bioreactors 12.3.3 Practical Cell Therapies 12.4 Tissue Engineering 12.4.1 Biomaterials to Tissue Engineering 12.4.2 Tissue Engineering and Regenerative Medicine Chapter 13 - Chemical Biology, Nanomedicine and Advanced Therapeutics 13.1 General Introduction 13.2 The Chemical Biology Approach to Gene Therapy 13.2.1 Nantechnology to Nanomedicine 13.2.2 Advanced Therapeutics - Gene Therapy 13.2.3 Gene Therapy Strategies 13.2.4 First Applications of Synthetic Nanoparticles in Gene Therapy Strategies 13.2.5 Improving on the Therapeutic Nucleic Acid APIs 13.2.6 The ABCD Nanoparticle Concept 13.2.7 Formation of ABC/ABCD Nanoparticles for in vivo and Clinical Use 13.2.8 Nanoparticles in Clinic 13.2.9 Desiging Future ABC/ABCD Nanoparticles 13.2.10 4Ss or Nanotechnology; Impact on 4 Ts of Delivery 13.2.11 Future Prospects for Gene Therapy Enabled by LNP Nanomedicine 13.3 Biophysical Characterisation of LNPs 13.3.1 Dynamic Light Scattering 13.3.2 Measuring Nanoparticulate Zeta-potentials 13.3.3 Measuring Nanoparticle Properties in Complex Solutions 13.4 Applications of LNPs with Small Molecule Drugs Chapter 14 - Chemical Biology and Advanced Diagnostics Leading to Precision Therapeutic Approaches 14.1 General Introduction 14.2 MRI Basic Principles Leading to Diagnostic Applications 14.2.1 Small Molecule Positive Contrast Agents 14.2.2 Imaging Nanoparticles 14.3 PET/CT and SPECT Fundamentals 14.3.1 Tracer Agents 14.3.2 Nanomolecular Tracer Agents 14.4 Understanding How to Control Nanoparticle Biodistribution Behaviour in vivo 14.4.1 Designing Future Theranostic ABC/ABCD Nanoparticles 14.5 Theranostics 14.5.1 Multimodal "hard" TNPs 14.5.2 Multimodal "soft" TNPs 14.5.3 Towards Precision Therapeutic Approaches for Treatment 14.5.4 Devising Precision Therapeutic Approaches for Treatment Chapter 15 - DNA Nanotechnology 15.1 Background 15.1.1 From Holliday Junctions to Double-Crossover and Paranemic DNA 15.1.2 Scaffolded DNA Origami 15.1.3 Single-Stranded Tile (SST) Assembly 15.2 Three-Dimensional DNA Nanostructures 15.3 Dynamic DNA Nanostructures 15.4 Biomedical Applicationns of DNA Nanostructures