Journal: iScience

Article Title: Monitoring GPCR conformation with GFP-inspired dyes

doi: 10.1016/j.isci.2024.110466

Figure Lengend Snippet: Structural and spectral changes of A 2A AR L225C -DyeC induced by various ligands (A) Schematic representation of structural changes caused by agonists and the allosteric modulator HMA in A 2A AR L225C -DyeC. Structures of the active and inactive A 2A AR are sketched from PDB IDs 6GDG and 3RFM , respectively. , Agonist binding results in an outward shift of the intracellular part of the TM6. The structural effects of the allosteric modulator HMA remain unknown. (B) Emission spectra of A 2A AR L225C -DyeC bound to antagonists (ZM241385 and SCH58261), agonists (NECA and adenosine), allosteric modulator (HMA), and in the apo state. (C) Variation of the integrated intensity of A 2A AR L225C -DyeC bound to different ligands. The integrated intensity is quantified as the area under the fluorescence emission spectrum from 430 to 700 nm. (D) Variation of the intensity ratio I 520 /I 460 for different ligands. Each condition in C and D was measured at least 9 times with protein from at least three independent purifications; each protein sample was mixed with the ligand independently. The data represent the mean ± SD. The protein concentration was maintained at 10 μM; all ligands were added at a saturating concentration of 100 μM. The significance level is given according to the ordinary one-way ANOVA with the post hoc Tukey HSD test: ∗∗ p < 0.005, ∗ p < 0.05, ns, not significant..

Article Snippet: Figure 4 Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations.

Techniques: Binding Assay, Fluorescence, Protein Concentration, Concentration Assay

Journal: iScience

Article Title: Monitoring GPCR conformation with GFP-inspired dyes

doi: 10.1016/j.isci.2024.110466

Figure Lengend Snippet: Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations. The A 2A AR L225C helices are labeled from TM1 to H8 (TM6 is colored in red), the G-protein binding site is labeled in the active state. (C and D) Positions of the dimethoxybenzene ring of the DyeC label in the active (C) and inactive (D) complexes throughout the unbiased (i.e., without any external forces applied) MD simulations shown every 0.1 ns as orange/yellow dots, respectively. Each system was simulated for 1,000 ns in two replicates. The positions of the lipid head groups are schematically indicated by the gray dotted line. (E) Autocorrelation functions (ACFs) calculated for a vector describing the DyeC label position in the unbiased simulations shown in (C) and (D). Higher values of ACF suggest slower reorientational dynamics of the label. Results for two replicates in the active/inactive states are shown in orange/yellow. (F) Correlation between fluorescence emission maximum of DyeC in different solvents and their partition coefficient, logP. The logP values were obtained from the PubChem/Chemeo databases , and provided in Table S1 . The inverse correlation implies that the translocation of DyeC into the region of the polar head groups of lipids, as observed in the simulations of the active state, leads to a red shift in the fluorescence emission maximum.

Article Snippet: Figure 4 Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations.

Techniques: Labeling, Protein Binding, Plasmid Preparation, Fluorescence, Translocation Assay

Journal: Journal of medicinal chemistry

Article Title: 2012 Philip S. Portoghese Medicinal Chemistry Lectureship: Structure-Based Approaches to Ligands for G Protein-Coupled Adenosine and P2Y Receptors, From Small Molecules to Nanoconjugates +

doi: 10.1021/jm400422s

Figure Lengend Snippet: A. Structures of selected AR ligands discussed in the text, including those that have been co-crystallized with the A 2A AR ( 1 , 2 , 5 , and 6 ). Dashed lines indicated the major H-bonding and π-bonding contacts between the receptor and ligand present in X-ray structures. Other ligands shown include pharmacological probes ( 4 and 9–12 ) and: advanced clinical candidates 3a and 3b for Parkinson’s disease; diagnostic agents for myocardial perfusion imaging 7 (approved and in trials for sickle cell anemia and ischemic conditions) and 8 (clinical candidate). B. The helical bundle of Family A GPCRs defines a cavity for the recognition of diverse ligands. The phospholipid bilayer is not shown. Figure courtesy of Stefano Costanzi (American Univ., Washington, DC). C. Historical progression of knowledge of the AR binding site(s). 19 , 20 , 37 , 39 , 41 , 45 Arrows in upper panels indicate the following interactions: Yellow, position of terminal amino group intended for covalent linking 37 ; orange, H-bonding interaction predicted between the exocyclic amine of adenine and a conserved Asn6.55 39 ; white, proximity of 3′ and 2′-hydroxyl groups with conserved His7.43 predicted using a neoceptor approach 41 . Lower panels: left, predicted docking of agonist 5 using the antagonist-bound X-ray structure 19 , 45 ; right, actual position of agonist 6 in X-ray structure. 20 , 24

Article Snippet: 19 , 20 A 2A AR complexes were crystallized by Heptares Therapeutics using receptors that are thermostabilized by systematic scanning mutagenesis (StaRs) include several other agonists and antagonists, including one with the antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl)oxy]phenyl]-1,3-dipropylxanthine 2 (xanthine amine congener, XAC).

Techniques: Diagnostic Assay, Imaging, Binding Assay