gtp hydrolysis generating amp  (Thermo Fisher)

Journal: Nature Communications

Article Title: Structure and function of a near fully-activated intermediate GPCR-Gαβγ complex

doi: 10.1038/s41467-025-56434-4

Figure Lengend Snippet: a GTP hydrolysis of the S4 mediated Gα s βγ as a function of inverse, partial, and full agonists, in reference to Gα s βγ alone (negative control) and the S5 mediated Gα s βγ (positive control). Data with error bars are presented as mean ± SEM of four independent experiments. Statistical analyses were performed using the ordinary one-way ANOVA compared to the R291A apo, *** p < 0.001, and **** p < 0.0001. b Time course of GTP hydrolysis of the S4 mediated Gα s βγ, in reference to Gα s βγ alone (negative control) and the S5 mediated Gα s βγ (positive control); the initial rate of each catalysis was calculated. The initial rate of each catalysis was calculated. Data are presented as mean values ± SD from three independent experiments ( n = 3). Source data are provided in the Source Data file. c Km values for S4 mediated Gα s βγ, in reference to Gα s βγ alone (negative control) and the S5 mediated Gα s βγ (positive control). Data are presented as mean values ± SD from three independent experiments ( n = 3). Source data are provided in the Source Data file. d BODIPY-FL-GDP binding assay. e BODIPY-FL-GTP binding assay. f BODIPY-FL-GTP-γ-S binding assay ( g ) GTP-GDP exchange rate comparison. h The structures of BODIPY-FL-GDP, BODIPY - FL-GTP, and BODIPY-FL-GTP-γ-S.

Article Snippet: All statistical tests such as GTP hydrolysis assessments were conducted using GraphPad Prism 9.0.

Techniques: Negative Control, Positive Control, Binding Assay, GTP Binding Assay, Comparison

Journal: eLife

Article Title: AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in castrate-resistant prostate cancer

doi: 10.7554/eLife.73396

Figure Lengend Snippet: ( A ) Wheat germ agglutinin (WGA) blocks AR-V7 nuclear import: we incubated cells with WGA, an inhibitor of nucleoporin-mediated nuclear transport and monitored GFP-AR-V7 localization by live-cell imaging. WGA kept AR-V7 in the cytoplasm in the presence of doxycycline suggesting that AR-V7 nuclear import requires active transport via the NPC. Representative confocal microscopy images were shown. Red: WGA labeling of membranes; Green: GFP-AR-V7. Nuc: nuclear AR-V; Cyto: cytoplasmic AR-V7. Scale bars represent 10 µm. ( B ) (related to ) HEK293T cells were transfected with plasmids encoding GFP-tagged AR-fl, AR-v567 and AR-V7 in the presence of the catalytic mutant Ran-GTP (mCherry-tagged RanQ69L). Nuclear accumulation of each AR variant was calculated. Representative confocal microscopy images (63× magnification) for each condition are shown. Solid arrow: cell with both cytoplasmic and nuclear proteins; arrowheads: cells with cytoplasmic protein; dashed arrow: cell with nuclear protein. Scale bars represent 10 µm. ( C ) Graphic display of % Nuclear AR across 30 individual cells per condition. Data represent mean ± SEM with n>10, p-value (**p<0.01, ****p<0.0001) was obtained using unpaired two-tailed t-test. ( D ) C4-2 cells with endogenous AR-fl stably expressing inducible GFP-AR-V7 were transfected with the catalytic mutant mCherry-tagged RanQ69L. Representative confocal microscopy images are shown. Cytoplasmic enrichment for AR-V7 is shown when the mutant Ran is co-expressed in the same cell. ( E ). 22RV1 cells with endogenous AR-fl and AR-V7 were transfected with the catalytic mutant mCherry-tagged RanQ69L. AR-V7 was detected by immunostaining with the RevMab antibody. Downregulation of endogenous AR-V7 protein was observed in cells co-expressing RanQ69L. Solid arrow: cell with cytoplasmic and nuclear AR-V7; dashed arrow: non-transfected cells with endogenous nuclear AR-V7 Dotted ellipses: cells transfected with mutant Ran where AR-V7 appears absent or downregulated. Scale bar, 10 µm. ( F ). PC3 cells were transfected with GFP-AR-fl or GFP-AR-fl-D-box mutant (A596T and S597T) and they were treated for 4 hr with 10 nM DHT. There was no effect of the D-box mutations on AR-fl nuclear import. Experiments were repeated at least twice.

Article Snippet: The construct containing mCherry-tagged GTP-hydrolysis defective Ran mutant, pmCherry-C1-RanQ69L (Addgene plasmid #30309), was a gift from Dr. Jay Brenman ( ) and used for live-cell imaging.

Techniques: Incubation, Live Cell Imaging, Confocal Microscopy, Labeling, Transfection, Mutagenesis, Variant Assay, Two Tailed Test, Stable Transfection, Expressing, Immunostaining

Journal: Nature Communications

Article Title: High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

doi: 10.1038/s41467-022-27967-9

Figure Lengend Snippet: a Top: (TI)-POST state of Ct 80S with eEF2-GDP shows the position of the conserved diphthamide (Dph) histidine modification at the apex of eEF2 domain IV. The monitoring adenines of the DC are marked in magenta. Bottom: The (TI)-POST state with tRNA •mRNA module (although still with non-hydrolyzable eEF2-‘GTP’). Dph is in same post-conformation as in our (TI)-POST state. Codons and associated tRNAs are color-coded. b The rotated ribosome acts as GAP for GTP hydrolysis in eEF2. Top: The full-rotated ribosome (40S body helix H5) pushes on the ordered switch 1 region (sw1) of eEF2 and places an intrinsic arginine finger. Bottom: Upon back-rotation (indicated by arrows), as observed in the (TI)-POST state of Ct 80S with eEF2-GDP-Mg 2+ , sw1 becomes disordered. c , d Switch 2 (sw2, green) conformations during the eEF2 switch cycle. In context of the PRE ribosome, the SRL is in tight contact with sw2 and the P-loop (light blue) of eEF2-‘GTP’ . Upon GTP hydrolysis and as detailed for (TI)-POST Ct 80S, the P-loop and sw2 relax and contacts are loose. Magnesium ions (magenta spheres) mediate SRL-eEF2 contacts. GDP is shown with its cryo-EM map (3 σ). e , f Switch 1 conformations during the eEF2 switch cycle. In the full-rotated state, the arginine finger within sw1 is placed on top of the scissile bond (finger-in) and the ribosome acts as GAP (not described there). Upon back-rotation of the 40S body (indicated by gray arrow), as seen in the (TI)-POST state of Ct 80S, sw1 becomes disordered and eEF2 relaxes in a relay system (distant domains II and III).

Article Snippet: GTP hydrolysis, and probably subsequent phosphate release, as shown very recently by detailed time-resolved cryo-EM studies for the bacterial system , , impacts on eEF2-ribosome interactions by modulating and loosening contact sites, as observed in our eEF2-GDP structure between the P-loop and the SRL.

Techniques: Modification, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: High-resolution structures of a thermophilic eukaryotic 80S ribosome reveal atomistic details of translocation

doi: 10.1038/s41467-022-27967-9

Figure Lengend Snippet: From left to right: Schematic for eukaryotic ribosomal states along a translocation cycle starting with the PRE state after peptide-bond formation. The m 1 acp 3 Ψ hypermodification (magenta bracket) within the 40S head, on top of position one of the anticodon in the wobble, helps as part of the wobble-seal in coupling ribosomal rotation to tRNA 2 •mRNA module movements between the classical A/A P/P and the A/P P/E hybrid states. Dph (highlighted in blue) within domain IV of eEF2-GTP acts as a doorstop ( pawl) in the A-site and the full-rotated state is stabilized. The full-rotated ribosome acts as GAP inducing GTP hydrolysis in eEF2, marking the transition to the translocated POST state. Back-rotation of the 40S head is uncoupled from the tRNA 2 •mRNA module and Dph acts as ‘ post-pawl’ in contact with the P-site. eEF2-GDP binding is weakened due to the internal relay and SRL-contact loosening and upon back-swiveling of the 40S head, eEF2-GDP is released. Translocation is complete in the P/P E/E state.

Article Snippet: GTP hydrolysis, and probably subsequent phosphate release, as shown very recently by detailed time-resolved cryo-EM studies for the bacterial system , , impacts on eEF2-ribosome interactions by modulating and loosening contact sites, as observed in our eEF2-GDP structure between the P-loop and the SRL.

Techniques: Translocation Assay, Binding Assay