Journal: Nature Structural & Molecular Biology

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

doi: 10.1038/s41594-026-01893-6

Figure Lengend Snippet: a , GTPγS–BDP-FL binding to nucleotide-free μOR–G i containing an unlabeled Gα i Δ6-90C/238C α-subunit was monitored by fluorescence dequenching for LFT (bottom) and BUP (top). Normalized kinetic spectra in the absence (gray) and presence (red) of 2 μM GDP fit to a single exponential function ( t , mean association time; dashed lines). b , Gα i α5 helix engaging the intracellular surface of μOR destabilizes the α1 helix and disrupts the interdomain interface (dashed outline) composed of αF, α1, L1 and the N-terminal portion of αA (PDB 1GP2 , blue; PDB 7T2H , orange). c , Schematic of main FRET states and proposed connection to α5 insertion and nucleotide (NT) binding. Full agonists bias the conformational equilibrium toward an open state (right) with reduced nucleotide affinity, while partial agonists shift the G protein toward a state with greater AHD closure (left). This shift increases the energy required for nucleotide release, thus reducing the likelihood of progressing to the nucleotide-free complex.

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

Techniques: Binding Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: YqeK is a Ap 4 A hydrolase. A , analytical size-exclusion chromatography of purified Bs YqeK and Gt YqeK. Both proteins elute at approximately 42 kDa, consistent with a homodimer. The grey shaded profile and calibration line represent the molecular-weight standards used to estimate the apparent molecular masses. The inset shows the SDS-PAGE of Bs YqeK and Gt YqeK. B , HPLC analysis of Ap 4 A hydrolysis by Bs YqeK with or without manganese, which promotes symmetrically converts of Ap 4 A into ADP. C , ITC analysis of Ap 4 A binding to B. subtilis YqeK. D , enzyme-kinetic analysis of B. subtilis YqeK mediated hydrolysis of Ap 4 A. A hyperbolic fit according to Michaelis-Menten was used to determine K m and V max . E , 5′-Ap 4 A-RNA decapping of Bs YqeK in vitro. Schematic representation of the 5′-Ap 4 A-RNA decapping reaction ( left ) and analysis of in vitro decapping ( right ) of WT Bs YqeK and Bs YqeK (H50A/D51A) mutant proteins and the positive control Ec ApaH using APB urea polyacrylamide gel electrophoresis.

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Size-exclusion Chromatography, Purification, Molecular Weight, SDS Page, Analysis, Binding Assay, In Vitro, Mutagenesis, Positive Control, Polyacrylamide Gel Electrophoresis, High Performance Liquid Chromatography, Isothermal Titration Calorimetry

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Relative Activity and Binding Affinity of YqeK Toward Ap 4 N Substrates

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Activity Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Quantification of intracellular Ap 4 N dinucleotides. Targeted QQQ LC–MS/MS analysis of Ap 4 A, Ap 4 G, Ap 4 C and Ap 4 U in B. subtilis WT 168, deletion (Δ yqeK ), overexpression (+ yqeK ) and complementation (Δ yqeK + yqeK ) strains at OD 600 0.5 ( A ), 1.0 ( B ), 3.0 ( C ) and overnight culture, OD 600 2.0 ( D ). Metabolite levels were normalized to dilution-corrected culture density and are shown as pmol/OD 600 .ml. Bars indicate mean values; points represent nine measurements from three biological replicates, with three technical replicates per clone. Statistical analyses were performed using GraphPad Prism. Technical triplicates were averaged within each biological replicate, and each growth stage was analyzed separately by two-way ANOVA followed by Dunnett’s multiple-comparisons test against Δ yqeK within each nucleotide group. Adjusted p values are indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Liquid Chromatography with Mass Spectroscopy, Analysis, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Structural insights into Gt YqeK. Overall structures of Gt YqeK ( A ) in the apo state, ( B ) in a complex with ADP, and ( C ) in a complex with the non-hydrolysable Ap 4 A analog Ap 2 Cp 2 A. ( D ) Detailed view of the deeply buried nucleotide-binding pocket in Gt YqeK. E, F , structural comparison of the ADP- and Ap 2 Cp 2 A-bound states reveal rearrangements of individual amino acid side chains in the phosphate-binding region and the second nucleotide-binding pocket.

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Binding Assay, Comparison, Docking Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Ap 4 A Hydrolysis and Binding Affinity by YqeK Variants

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Binding Assay