This book summarizes the progress made to functional immobilize G protein-coupled receptors (GPCRs) through site-specific or orientated recognition in both non-covalent and covalent manners. The last decade is the dawn of the “post-structural biology” era for G protein-coupled receptor research. As an emerging approach for state-of-the-art immobilization, this book discusses efforts to explore the elegance of naturally-occurring biochemical reactions by using their high specificity and robust reactivity in the complex system, such as site-specific conjugation by covalent recognition between enzymes and their substrates. With the perspective of protein-drug interactions, this book also reviews the applications of protein immobilization, with an emphasis on G protein-coupled receptors, in drug discovery and protein-ligand interaction analysis. In addition, the merits, opportunities and disadvantages are analyzed for different immobilization methods, and a perspective for future directions is presented. Given its scope, this book appeals to a broad readership, particularly researchers engaged in the field of analytical chemistry, bioconjugate chemistry, and chemical biology, and other related field, as well as teachers of relevant majors in colleges and universities.
Author(s): Xinfeng Zhao, Qian Li, Jing Wang, Qi Liang, Jia Quan
Series: SpringerBriefs in Molecular Science
Publisher: Springer
Year: 2023
Language: English
Pages: 99
City: Singapore
Preface
Perspectives
Contents
About the Authors
1 G Protein-Coupled Receptors
1.1 GPCR Biology
1.2 GPCR Structures
1.3 GPCR Pharmacology
1.3.1 G Protein-Coupled Receptor Conformations
1.3.2 G Protein-Coupled Receptor Signaling Pathway
References
2 Purification of G Protein-Coupled Receptors
2.1 GPCR Solubilization and Stabilization
2.1.1 Detergents
2.1.2 Styrene Maleic Acid Lipid Particles
2.2 Purification Techniques for GPCRs
2.3 GPCR Purification from Native Tissues
2.4 Heterologous Expression of GPCRs
2.4.1 GPCRs Expressed in E. coli
2.4.2 GPCRs Expressed in Yeast
2.4.3 GPCRs Expressed in Insect Cell-Line
2.4.4 GPCRs Expressed in Mammalian Cell-Line
2.4.5 GPCRs Expressed in Drosophila melanogaster
References
3 Oriented Immobilization of G Protein-Coupled Receptors
3.1 Affinity-Based Non-Covalent Immobilization
3.1.1 Histidine Tag
3.1.2 Peptide Tags
3.1.3 Biotin–(Strept) Avidin Interaction
3.1.4 DNA-Mediated Immobilization
3.1.5 Glutathione-S-Transferase (GST) Tag
3.2 Site-Specific Covalent Immobilization Through Bioorthogonal Chemistry
3.2.1 Staudinger Ligation
3.2.2 Diels–Alder Reaction
3.2.3 Oxime Ligation
3.2.4 Photochemical Thiol-Ene Reaction
3.2.5 Copper(I)-Catalyzed Azide-Alkyne Cycloaddition
3.3 Biologically Mediated Site-Specific Immobilization
3.3.1 Enzyme-Catalyzed Immobilization
3.3.2 Self-Labelling Active Enzyme
3.3.3 Native Chemical Ligation
References
4 Key Biochemical Aspects of Drug-Target Interactions
4.1 Binding Affinity and Kinetics
4.2 Dynamic Conformational Ensembles of Drug Targets
4.3 Drug Resistance Time
4.4 Rebinding
4.5 Binding Thermodynamics
4.6 Ligand Efficiency
References
5 Immobilized GPCRs in Drug-Receptor Interaction Analysis
5.1 Labeled Drug-Receptor Binding Assays
5.1.1 Radioligand Binding Assays
5.1.2 Fluorescent Ligand Binding Assays
5.2 Label-Free Drug-Receptor Binding Assays
5.2.1 Surface Plasmon Resonance (SPR)
5.2.2 Resonant Waveguide Grating (RWG)
5.2.3 Interferometry Biosensor
5.2.4 Atom Force Microscopy
5.2.5 Receptor Chromatography (Column-Based Assays)
References
6 Immobilized GPCRs in Compound Screening
6.1 Screening of Compound Libraries
6.1.1 Sensor-Based Assays
6.1.2 GPCR Microarrays
6.1.3 Affinity Chromatography
6.2 Screening of Natural Product Extracts
References
