Journal: The EMBO Journal

Article Title: K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins

doi: 10.1038/s44318-024-00146-w

Figure Lengend Snippet: ( A ) Superimposition of five representative complex conformations from the ensemble clusters for the K128-ubiquitinated NRAS/ RASA1 GAP complex. ( B ) The interaction energies of RASA1 GAP with non-ubiquitinated NRAS and NRAS ubiquitinated at K128. The interaction energies were calculated over the entire trajectories and then averaged. Data represented as mean ± SD. N = 1250 snapshots. ( C ) Chemical ubiquitination of NRAS-C118S/ K128C mutant and purification of monoubiquitinated NRAS by size-exclusion chromatography. Proteins were separated by SDS-PAGE under non–reducing conditions or in the presence of a reducing agent, tris(2-carboxyethyl) phosphine (TCEP), and stained by Coomassie blue. ( D ) In vitro Co-IP of non-conjugated or ubiquitin-conjugated NRAS-K128C and RAF1. Equimolar amounts of NRAS-K128C/ Ubiquitin-G76C complex or NRAS-K128C mutant were incubated with V5-tagged RAF1 in the presence of GDP or GTPγS. ( E ) In vitro Co-IP of non-conjugated or ubiquitin-conjugated NRAS-K128C and SOS1. Equimolar amounts of NRAS-K128C-Ubiquitin-G76C complex or NRAS-K128C mutant were incubated with equal amounts of the GDP/GTP exchange domain of SOS1 in the presence of GDP. ( F ) In vitro Co-IP of non-conjugated or ubiquitin-conjugated NRAS-K128C and RASA1. Equimolar amounts of the indicated NRAS proteins were incubated with equal amounts of RASA1 GAP in the presence of GTPγS. ( G ) RASA1-mediated GTPase activity of ubiquitinated and non-ubiquitinated NRAS normalized to intrinsic GTPase activity. Equimolar amounts of the indicated NRAS proteins were mixed with the GAP domain of RASA1. GTP hydrolysis reaction was initiated by the addition of GTP. Data were present as mean ± s.e.m. N = 3 technical replicates in independent experiments. P -value was calculated by a two-sided t -test. ( H ) wt-RAS and ubiquitination-deficient RAS mutant were co-immunoprecipitated with GST-tagged RASA1 GAP using anti-Flag resin followed by immunoblotting with the indicated antibodies. ( I ) Superimposition of five representative complex conformations from the ensemble clusters for the K128-ubiquitinated KRAS/ NF1 GRD complex. ( J ) The interaction energies of NF1 GRD with non-ubiquitinated KRAS and KRAS ubiquitinated at K128. Data represented as mean ± SD. N = 1250 snapshots. ( K ) The indicated RAS proteins were co-immunoprecipitated with GST-tagged NF1 GRD using anti-Flag resin, followed by immunoblotting with the indicated antibodies. .

Article Snippet: For in vitro immunoprecipitation experiments, NRAS was loaded with GDP or GTPγS by mixing RAS with a 10x stochiometric excess of GDP or GTPγS (Jena Bioscience) in a buffer containing an excess (20 mM) of EDTA.

Techniques: Ubiquitin Proteomics, Mutagenesis, Purification, Size-exclusion Chromatography, SDS Page, Staining, In Vitro, Co-Immunoprecipitation Assay, Incubation, Activity Assay, Immunoprecipitation, Western Blot

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