Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Use of Fluorescence Indicators in Receptor Ligands
Fluorescence techniques can provide insights into the environment of fluorescence indicators incorporated within a ligand as it is bound to its receptor. Fluorescence indicators of different sizes and chemical characteristics can provide insights into the nature of the binding environment, the surrounding structures, and even into conformational changes associated with receptor activation. Methods for determining fluorescence spectral analysis, fluorescence quenching, fluorescence anisotropy, fluorescence lifetimes, and red edge excitation shifts of the ligand probes are described. The applications of these techniques to t...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Monitoring G Protein Activation in Cells with BRET
Live-cell assays based on fluorescence and luminescence are now indispensable tools for the study of G protein signaling. Assays based on fluorescence and bioluminescence resonance energy transfer (FRET and BRET) have been particularly valuable for monitoring changes in second messengers, protein–protein interactions, and protein conformation. Here, we describe a BRET assay that monitors the release of free Gβγ dimers after activation of heterotrimers containing Gα subunits from all four G protein subfamilies. This assay provides useful kinetic and pharmacological information with reasonably high thr...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Approaches to Characterize and Quantify Oligomerization of GPCRs
Fluorescence resonance energy transfer (FRET) is an approach widely used to detect protein–protein interactions in live cells. This approach is based on the sensitization of an “acceptor” molecule by the energy transfer from a “donor” when there is an overlap between the emission spectrum of the “donor” and the excitation spectrum of the “acceptor” and close proximity between the two fluorophore species (in the region of 8 nm). Various methods exist to quantify FRET signals: here, we describe the application of homogeneous time-resolved FRET (htrFRET) combined with Tag-...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Quantitative Multi-color Detection Strategies for Bioorthogonally Labeled GPCRs
We describe multiple bioorthogonal approaches to label G protein-coupled receptors (GPCRs) heterologously expressed in mammalian cells. The use of genetically encoded unnatural amino acids as bioorthogonal tags results in receptors that are expressed at lower levels than even their low abundance wild-type counterparts. Therefore, reproducible and sensitive quantification of the labeled GPCRs is extremely important and conventional methods are simply not sufficiently accurate and precise. Silver stains lack reproducibility, spectroscopic methods using fluorescent ligands are limited to quantifying only functional receptor m...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Single-Molecule Fluorescence Microscopy for the Analysis of Fast Receptor Dynamics
Assessing the dynamics of individual membrane proteins in living cells is a powerful approach to investigate their assembly, mobility, and function. Here, we describe how to image single G protein-coupled receptors (GPCRs), both in the active and inactive state. This is achieved by combining labeling of GPCRs with bright organic fluorophores and fluorescent imaging by total internal reflection fluorescence microscopy. Using this method, individual tracks of single molecules can be analyzed in parallel with high spatial precision and with frame rates up to 50/s. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Nuts and Bolts of CF3 and CH3 NMR Toward the Understanding of Conformational Exchange of GPCRs
With the advent of efficient protein expression and functional purification protocols, it is now possible to reconstitute many G protein-coupled receptors (GPCRs) in detergent micelles at concentrations of 25 μM or more. Such concentrations are sufficient for studies of conformational states and dynamics relating to function and the mechanism of activation of GPCRs, using solution state NMR. In particular, methyl spectroscopy, in the form of one-dimensional 19F NMR or two-dimensional (1H,13C) NMR, provides high fidelity spectra which reveal detailed features associated with conformational states and their lifetimes, as ...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy
While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (<150 kDa) complexes between GPCRs and their partner proteins. Negative stain EM, whereby the biological samples are embedded in a thin layer of heavy metal solution, is a well-established alternative technique that provides the enhanced contrast needed to visualize small macromolecular complexes. This methodology can offer a simple and powerful tool for the rapid evaluation of sample characteristics, such as homogeneity or ol...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Purification and Crystallization of a Thermostabilized Agonist-Bound Conformation of the Human Adenosine A2A Receptor
Crystallization of G protein-coupled receptors (GPCRs) is successful due to the development of generic protein engineering strategies, which has resulted in the structure determination of more than 25 GPCRs, including representatives from class A, B, C, and F. Most of the X-ray structures available correspond to an inactive conformation of the receptor bound to an antagonist. Only a few high-resolution structures of agonist-bound conformations of GPCRs have been determined over the last 6 years. Here, we describe the purification and crystallization protocols of a thermostabilized agonist-bound conformation of the human ad...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Interaction Fingerprints and Their Applications to Identify Hot Spots
Binding recognition is in the core of how nature controls processes in living cells, how enzyme–substrate binding leads to catalysis and how drugs modulate enzymes and receptors to convey a desirable physiological response. Thus, understanding binding recognition in a systematic manner is paramount, not only to understand biological processes but also to be able to design and discover new bioactive compounds. One such way to analyze binding interactions is through the development of binding interaction fingerprints. Here, we present the methodology to develop interaction fingerprints with three different software pla...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Purification of Stabilized GPCRs for Structural and Biophysical Analyses
G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor t...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Experiment-Guided Molecular Modeling of Protein–Protein Complexes Involving GPCRs
Experimental structure determination for G protein-coupled receptors (GPCRs) and especially their complexes with protein and peptide ligands is at its infancy. In the absence of complex structures, molecular modeling and docking play a large role not only by providing a proper 3D context for interpretation of biochemical and biophysical data, but also by prospectively guiding experiments. Experimentally confirmed restraints may help improve the accuracy and information content of the computational models. Here we present a hybrid molecular modeling protocol that integrates heterogeneous experimental data with force field-b...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
The Dynamic Process of Drug–GPCR Binding at Either Orthosteric or Allosteric Sites Evaluated by Metadynamics
Major advances in G Protein-Coupled Receptor (GPCR) structural biology over the past few years have yielded a significant number of high-resolution crystal structures for several different receptor subtypes. This dramatic increase in GPCR structural information has underscored the use of automated docking algorithms for the discovery of novel ligands that can eventually be developed into improved therapeutics. However, these algorithms are often unable to discriminate between different, yet energetically similar, poses because of their relatively simple scoring functions. Here, we describe a metadynamics-based approach to ...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Docking and Virtual Screening Strategies for GPCR Drug Discovery
Progress in structure determination of G protein-coupled receptors (GPCRs) has made it possible to apply structure-based drug design (SBDD) methods to this pharmaceutically important target class. The quality of GPCR structures available for SBDD projects fall on a spectrum ranging from high resolution crystal structures (<2 Å), where all water molecules in the binding pocket are resolved, to lower resolution (>3 Å) where some protein residues are not resolved, and finally to homology models that are built using distantly related templates. Each GPCR project involves a distinct set of opportunities and ch...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Radioligand Binding Assay for an Exon 11-Associated Mu Opioid Receptor Target
Receptor binding provides a valuable approach for characterization of drugs and their receptors. There are three major families of opioid receptors: mu, delta, and kappa. Highly selective radioligands are available for all three classes of traditional receptors. Of the three, the mu receptor undergoes extensive alternative splicing, generating a number of traditional mu receptor subtypes as well as a nontraditional, truncated set of variants associated with exon 11. These exon 11-associated truncated variants are not readily labeled with current radioligands. Here we describe the synthesis of a radioiodinated ligand suitab...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
High-Throughput Screening for Allosteric Modulators of GPCRs
We describe several HTS protocols designed for the identification of GPCR ligands, with a particular focus on the identification of allosteric modulators. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
