Journal: bioRxiv

Article Title: Ligand-dependent G protein dynamics underlying opioid signaling efficacy

doi: 10.1101/2025.06.30.662233

Figure Lengend Snippet: a , Site-specific labeling: SDS-PAGE analysis of purified Gα i Δ6 ‘cys-less’ background with and without engineered cysteines (R90C/E238C) labeled with either LD555 (left) or JFX673 (right) visualized using fluorescence imaging (top; 532 nm or 633 nm illumination), with Coomassie staining (bottom) as a gel-loading control. b , Representative room-temperature size exclusion chromatography (SEC) profiles of buprenorphine-bound μOR alone (dashed line) and as a ternary complex with dye-labeled G i heterotrimer (absorption in gray; LD555 fluorescence in green). c , Specificity of M1 fab-mediated immobilization: Uptake of 200 pM labeled μOR-G i onto passivated quartz slides coated with M1 fab fragment (blue; specific) and without M1 fab (gray; non-specific) recorded using 100 ms TIRF imaging at low laser power (1 mW at the source) to prevent photobleaching. d , Histogram of immobilized FRET particles (mean ± 95% c.i., n = 3 movies of the same sample) remaining after injection of buffer (vehicle) or 10 μM GTPγS into the imaging chamber shows GTPγS binding and functional dissociation of μOR-G i on the surface.

Article Snippet: Binding of Bodipy conjugated GTPγS (GTPγS-BDP-FL, Jena Bioscience) to nucleotide-free μOR-G i complexes was measured as previously described ( 91 ).

Techniques: Labeling, SDS Page, Purification, Fluorescence, Imaging, Staining, Control, Size-exclusion Chromatography, Injection, Binding Assay, Functional Assay

Journal: bioRxiv

Article Title: Ligand-dependent G protein dynamics underlying opioid signaling efficacy

doi: 10.1101/2025.06.30.662233

Figure Lengend Snippet: a , GTPγS, but GMP with PPi, dissociates G protein from surface-tethered μOR. Error bars, mean ± 95% c.i. (n = 3-6 movies of the same sample) of FRET particles remaining after delivery of 10 μM nucleotide or buffer (vehicle); p = 0.23 (ns), two-sided, unpaired t-test. b , Schematic of single-molecule GMP·PPi titration experiments. c , Population FRET efficiency histograms (symbols) with b-spline fits (lines) for DAMGO-bound μOR-G i in the presence of increasing concentrations of GMP·PPi. d , Apparent EC 50 values measured from GMP·PPi-induced ensemble average low-FRET occupancy for DAMGO (blue) and morphine (gray); error bars, s.d. of 100 bootstrap samples of the FRET traces (3,341 molecules total across all conditions). Lines are fits to dose response functions (Hill slope = 1.0). e,f , Transition density plot ( e ) for DAMGO with 10 μM GMP·PPi (scale bar, 10 −3 transitions per bin per second; n t total transitions) and contour plot of synchronized high-to-low transitions ( f ) showing a short-lived dwell in a mid-FRET state (yellow box, width ≈ one frame) (cf. Extended Data Fig. 6e).

Article Snippet: Binding of Bodipy conjugated GTPγS (GTPγS-BDP-FL, Jena Bioscience) to nucleotide-free μOR-G i complexes was measured as previously described ( 91 ).

Techniques: Titration