mant atpγs  (Jena Bioscience)

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet: (A) Chemical structure of ′(3′)-O-(N-methyl-anthraniloyl) ATPγS (mant-ATPγS). (B) ATPγS is not hydrolyzed by nsP2. Under the conditions tested, 50 nM nsP2 converted ∼80% of 1 mM ATP to ADP within 30 minutes, whereas no detectable hydrolysis of 1 mM ATPγS was observed after 120 minutes. No luminescence signal was detected in reactions containing ATP or ATPγS in the absence of enzyme (data not shown). (C) Representative tryptophan to mant FRET emission spectra collected using excitation at 80 nm. nsP2 alone (1 μM) exhibits an emission peak at 350 nm, whereas mant-ATPγS alone (10 μM) shows weak emission at 445 nm under 280-nm excitation. Addition of mant-ATPγS to nsP2 products an increase in 445-nm emission, consistent with FRET arising from formation of the nsP2·mant-ATPγS complex. Data in panels D-G were generated by subtracting mant-ATPγS-only emission at 445 nm from spectra collected in the presence of nsP2. (D) Direct binding of mant-ATPγS to nsP2. ns2P (0. 25 μM) was titrated with 0.002 5 μM mant-ATPγS. Data represent mean ± SD ( n = 3). (E-G) Competitive binding experiments. nsP2 (0. 5 μM) was incubated with 0.1 μM mant-ATPγS and increasing concentrations of unlabeled competitor. ATPγS (E; 0-10 μM), ADP (F; 0-9 mM), or inorganic phosphate (Pi) and tripolyphosphate (TPP) (G; 0-40 mM) were added as indicated. Fluorescence data in panels E and F were normalized to percent relative fluorescence, with the signal in the absence of competitor defined as 100%. Data were fit by nonlinear regression, and IC₅₀ values were converted to inhibition constants ( K i ) using the Cheng-Prusoff equation.

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Generated, Binding Assay, Incubation, Fluorescence, Inhibition

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet: (A) Exp rimental design for ATPγS association kinetics. nsP2 was rapidly mixed with mant-ATPγS under stopped-flow conditions, and binding was monitored by tryptophan-to-mant FRET. (B) ATPγS association kinetics. Representative fluorescence time courses following rapid mixing of mant-ATPγS (0.1 μM) with increasing concentrations of nsP2 (0.5-3 μM). (C) Observed rate constants ( k obs ) extracted from single-phase fits to the association traces in panel B were replotted as a function of nsP2 concentration (n = 3 independent experiments). Linear regression was used to determine the second-order association rate constant ( k ₒₙ). (D) Experimental design for ATPγS dissociation kinetics. Pre-formed nsP2·mant-ATPγS complexes were rapidly mixed with excess unlabeled ATPγS to initiatw ligand displacement. (E) ATPγS dissociation kinetics. Time-dependent loss of sensitized Mant fluorescence following competition with unlabeled ATPγS. Traces were fit to a two-phase exponential decay, revealing fast and slow dissociation components ( k off,fast and k off,slow ). (F) Experimental design for ATP dissociation in the presence of inhibitor. Pre-formed nsP2·mant-ATP complexes were rapidly mixed with excess unlabeled ATP in the presence of the nsP2 inhibitor RA-NSP2- (5 μM). (G) ATP dissociation kinetics in the presence of inhibitor. Representative fluorescence decay trace fit to a single-phase exponential model, yielding the apparent ATP dissociation rate constant ( k off ).

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Binding Assay, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet:

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Binding Assay, Fluorescence, Concentration Assay

Journal: Nature Communications

Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

doi: 10.1038/ncomms9520

Figure Lengend Snippet: ( a ) The number of m-ATPγS that bound to PAN (90 nM) was determined by rapid separation of bound nucleotide from free nucleotide using 100 μl spin columns at two different concentrations of ATPγS: 10 and 200 μM. Ten micromolar saturates only the two high-affinity sites, and 200 μM allows near saturation of the high- and low-affinity sites (ATP and ADP sites; ref. ). The number of bound nucleotides per PAN hexamer was calculated for WT and each arginine mutant as labelled. Data are means of four independent experiments ±s.d. ( b ) Emission spectra of m-ATP as in , but with PAN-R328/331A (1 μM). Quantifications are presented on . ( c , d ) The number of m-ATPγS-bound nucleotides to the labelled PAN variant was calculated as in a at increasing nucleotide concentrations to generate a binding curve. [PAN] was 200 nM and thus the free ligand bind approximation is not met here and thus the K -value is expressed as Kobs as it does not accurately quantify affinity. Representative data are presented from three independent experiments ±s.d.

Article Snippet: Mant - ATPγS and Mant-ADP were purchased from Jena Bioscience.

Techniques: Mutagenesis, Variant Assay, Binding Assay

Journal: Nature Communications

Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

doi: 10.1038/ncomms9520

Figure Lengend Snippet: ( a ) Fluorescence polarization was used to monitor the binding of GFP–ssrA (0.08 μM) to PAN (0.12 μM) or its arginine mutants in the presence of 1 mM ADP (negative control) or 1 mM ATPγS. ( b ) Gate opening in the 20S proteasome (20 nM) by PAN WT, or its mutants (80 nM), was monitored with the LFP peptide hydrolysis in the presence of 10 μM ATPγS. ‘No PAN' is 20S (archaeal) alone. ( c ) Gate opening in the 20S proteasome (20 nM) as a function of increasing concentration of WT-PAN and arginine mutants. ( d ) The gate-opening assay by the WT-PAN (10 nM) as in b but also in the presence of the other indicated PAN mutants (10 nM) to determine whether the mutants can compete with WT for binding to the 20S. All data are representative experiments and are the means of three independent measurements ±s.d.

Article Snippet: Mant - ATPγS and Mant-ADP were purchased from Jena Bioscience.

Techniques: Fluorescence, Binding Assay, Negative Control, Concentration Assay

Journal: Nature Communications

Article Title: ATP binding to neighbouring subunits and intersubunit allosteric coupling underlie proteasomal ATPase function

doi: 10.1038/ncomms9520

Figure Lengend Snippet: ( a ) Pre-steady-state dissociation of the prebound m-ADP (150 nM) from WT-PAN (150 nM) was monitored by stopped-flow at 37 °C. Saturating amounts ADP (2 mM) were used to compete off the m-ADP. The residuals from fitting the raw data with single- or double-exponential decay models are shown (right). ( b ) The half-life ( T 1/2 ) of the bound m-ADP to WT-PAN and the arginine mutants for the double-decay model is presented, showing both fast and slow rates. ( c ) Pre-steady state dissociation of prebound m-ATPγS (1 μM) from WT-PAN (0.5 μM) was monitored as in a . Saturating amounts ADP (4 mM) were used to compete off the m-ATPγS. Residuals for the single- and double-decay models are shown (right). The determined half-life for both fast and slow rates for m-ATPγS are shown in the inset (double-decay model).

Article Snippet: Mant - ATPγS and Mant-ADP were purchased from Jena Bioscience.

Techniques: