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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers G protein coupled r… Read more
LIMITED OFFER
Immediately download your ebook while waiting for your print delivery. No promo code needed.
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers G protein coupled receptors, and includes chapters on such topics as GPCR modelling, interactions with other molecules, virtual screening and GPCR activation.
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
Contributors
Preface
Methods in Enzymology
Chapter One. The Protein Local Optimization Program and G-Protein-Coupled Receptors: Loop Restoration and Applications to Homology Modeling
1 Introduction
2 PLOP Performance for Loop Prediction
3 PLOP and GPCRs
4 Loop Prediction with the Single-Residue Library
5 Loop Prediction with the Dipeptide Library
6 Hierarchical Loop Prediction with Surrounding Side Chain Optimization
7 Additional Sampling Methods
8 VSGB 2.0: The Newest Energy Model Incorporated into PLOP
9 Conclusions
References
Chapter Two. Modeling Active GPCR Conformations
1 Introduction
2 General Approaches
3 Agonist-Induced Activation
4 Simulation Methods
5 Microswitches
6 Biophysical Restraints
7 Virtual Screening
8 Class B GPCRs
References
Chapter Three. Molecular Modeling of Adenosine Receptors
1 Introduction
2 Adenosine A2a Receptor
3 Receptor Modeling
4 Summary
References
Chapter Four. Predicting G-Protein-Coupled Receptors Families Using Different Physiochemical Properties and Pseudo Amino Acid Composition
1 Introduction
2 GPCRs Prediction
3 Performance Evaluation of GPCR Predictor
4 Webserver for GPCR Predictor
5 Concluding Remarks
References
Chapter Five. Computational Design and Experimental Characterization of GPCR Segment Models
1 Introduction
2 Design of GPCR Segment Models
3 Experimental Characterization of GPCR Segment Models
4 Integration of GPCR Segment Models into GPCR Homology Models
5 Summary
References
Chapter Six. G-Protein-Coupled Receptor Interaction with Small GTPases
1 Introduction
2 Assays for GPCR Interaction with Small GTPases
3 Function of GPCR–Small GTPase Interaction
References
Chapter Seven. Quantification of Receptor Tyrosine Kinase Activation and Transactivation by G-Protein-Coupled Receptors Using Spatial Intensity Distribution Analysis (SpIDA)
1 Introduction
2 Theory of Spatial Intensity Distribution Analysis
3 Approach Validation
4 Procedure for SpIDA
5 Conclusions
References
Chapter Eight. NMR Methods for Detection of Small Molecule Binding to RGS4
1 Introduction
2 Limitations and Challenges of NMR Experiments on RGS Proteins
3 Construct Selection for NMR Studies with RGS4
4 Expression and Purification of 15N-Labeled Δ51 RGS4
5 Setting Up a Protein-Based NMR Fragment Screen Targeting Δ51 RGS4
6 Alternative Ligand-Based Approach Using Saturation Transfer Difference for Small Molecule Studies with Unlabeled RGS
7 Conclusion
References
Chapter Nine. Group II Activators of G-protein Signaling: Monitoring the Interaction of Ga with the G-protein Regulatory Motif in the Intact Cell
1 Introduction
2 Generation of Probes for Measurement of GPR–Gαi Interaction in Cells by BRET
3 Expression of Donor and Acceptor in Cells
4 Analysis of GPR–Gαi1 Interaction by BRET
5 Regulation of the GPR–Gαi Complex by a G-protein-Coupled Receptor
6 Analysis of GPR–Gαi1 Interaction in Subcellular Fractions by BRET
7 Summary
References
Chapter Ten. Homogeneous Time-Resolved Fluorescence Assay to Probe Folded G Protein-Coupled Receptors
1 Introduction
2 Solid-Phase Labeling of IgG with Europium Cryptate
3 HTRF Assay Standards and Signal Analysis
4 Applications of GPCR HTRF Assay
5 Conclusion
References
Chapter Eleven. cAMP Biosensors Applied in Molecular Pharmacological Studies of G Protein-Coupled Receptors
1 Introduction
2 Generation of an Epac1-Based cAMP Biosensor Cell Line with an Optimized Fluorescent Protein FRET Pair
3 cAMP Biosensor Characterization
4 Assay for Studying GPCR Signaling as Exemplified by Gs-Coupled β2-Adrenergic Receptor Activation
5 Modifications to the General cAMP Kinetics Assay
References
Chapter Twelve. Rhodopsin–Lipid Interactions Studied by NMR
1 Introduction
2 Reconstitution of GPCR into Single, Tubular Bilayers Using Nanoporous Substrates
3 Nuclear Magnetic Resonance
4 Conclusions
References
Chapter Thirteen. Biasing the Parathyroid Hormone Receptor: Relating In Vitro Ligand Efficacy to In Vivo Biological Activity
1 Introduction
2 Determining the Relative Activity of PTH1R Ligands
3 Discussion
References
Chapter Fourteen. Application of Monte Carlo-Based Receptor Ensemble Docking to Virtual Screening for GPCR Ligands
1 Introduction
2 Description of the Methodology
3 A Practical Example: Application of Monte Carlo-Based RED to a Controlled Retrospective Virtual Screening for β2 Adrenergic Receptor Ligands
4 Final Considerations
References
Chapter Fifteen. From Heptahelical Bundle to Hits from the Haystack
1 From Heptahelical Bundles to Hits from the Haystack: Structural Molds for Virtual Screening
2 Shaping Structures, Carving Cavities: A Customized Structure-Based GPCR Modeling Workflow
3 In Silico Screening Success Stories: From GPCR Structure to Novel GPCR Ligands
4 Navigating Novel GPCR–Ligand Interaction Space
References
Chapter Sixteen. Kinetics and Dynamics in the G Protein-Coupled Receptor Signaling Cascade
1 Introduction
2 FRET Assays to Measure Reaction Kinetics in GPCR Signaling Cascades
3 Dynamic of Receptor–Protein Interactions
4 ICCS to Determine GPCR Dynamics
References
Chapter Seventeen. Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy of G Protein-Coupled Receptors
1 Introduction
2 Large-Scale GPCR Expression in HEK293 Cells
3 Reconstitution of GPCRs
4 MAS NMR Spectroscopy of GPCRs
References
Chapter Eighteen. Posttranslation Modification of G Protein-Coupled Receptor in Relationship to Biased Agonism
Abbreviations
1 Introduction
2 Posttranslation Modification of GPCR with Biased Signaling
3 Membrane Distribution of GPCR with Biased Signaling
4 Possible Interaction Between These Three Methods
5 Future Direction of Biased Signaling
References
Chapter Nineteen. Protein S-Nitrosylation Measurement
1 Introduction
2 Biotin Switch Technique
3 Fluorescence Switch Technique
4 His-Tag Switch Technique
5 In Situ BST
6 SNO-RAC
7 Mass Spectrometry Analysis
8 NO Detection Methods
9 S—NO Antibody
10 Summary
References
Author Index
Subject Index
PC