Journal: bioRxiv

Article Title: A nucleotide-triggered molecular switch orchestrating septin polymerization

doi: 10.64898/2026.05.07.723466

Figure Lengend Snippet: a , Nucleotide content of purified monomeric G-domains (Cdc10, Cdc3, Cdc12, Cdc11, Shs1) and co-expressed G-interface dimers (Cdc10-Cdc3, Cdc12-Cdc11, Cdc12-Shs1). Left: representative HPLC chromatograms from heat-denatured samples. Right: quantified nucleotide occupancy in dimers (arithmetic mean ± s.d., n = 3 independent experiments). Monomers are nucleotide-free; dimers are nucleotide-bound. b , Apparent GTP hydrolysis rates ( kcat,app ) measured by a malachite green assay at 100 µM GTP. Data are arithmetic means + symmetric 95% confidence intervals (CIs). Each data point represents the average of 2–12 technical replicates from independent experiments (Cdc10 n = 4, Cdc12 n = 4, Cdc3 n = 3, Cdc11 n = 3, Shs1 n = 3, Cdc10-Cdc3 n = 3 (MgCl2) or 4 (EDTA), Cdc12-Cdc11 n = 3 (MgCl2) or 4 (EDTA), Cdc12-Shs1 n = 4). For MBP, six replicates from the same snap-frozen protein preparation were measured on different occasions. Constructs with detectable activity are indicated (+). Significance brackets indicate results from one-way ANOVA with Tukey’s post hoc test. c , Representative stopped-flow fluorescence traces of EDA-GTP-BDP-FL association and dissociation with monomeric Cdc10. Global non-linear fits are shown in neon green. Kinetic parameters ( kon , koff , KD ) are geometric means with asymmetric 95% CIs from five independent experiments (association was measured at 1.0–4.2 µM Cdc10 (three preparations) or 1.0–3.1 µM (two preparations)). d , Investigation of dimer integrity via analytical SEC. Guanine nucleotide binding stabilizes G-interfaces. Co-expressed pairs (Cdc10-Cdc3, Cdc12-Cdc11, Cdc12-Shs1) form stable nucleotide-bound dimers. Incubation at 30 °C promotes dissociation, which is prevented by the addition of GTP and GDP. Phosphatase treatment (rSAP) amplifies dimer disruption.

Article Snippet: For association kinetics, constructs #1 and #2 were measured under pseudo-first-order conditions at final protein concentrations between 1.0 and 4.2 μM in the presence of 0.08 μM EDA-GTP-BDP-FL (Jena Bioscience), whereas construct #4 was assayed at final protein concentrations of 21 to 104 μM with 0.8 μM EDA-GTP-BDP-FL.

Techniques: Purification, Malachite Green Assay, Construct, Activity Assay, Fluorescence, Binding Assay, Incubation, Disruption

Journal: bioRxiv

Article Title: A nucleotide-triggered molecular switch orchestrating septin polymerization

doi: 10.64898/2026.05.07.723466

Figure Lengend Snippet: a , Nucleotide-content analysis of purified, heat-denatured monomeric G-domains after incubation with 3 mM GTP and GDP followed by desalting. No stably bound nucleotide was detected. A protein-free desalting control is shown for comparison. b , GTP hydrolysis by monomeric Cdc10 and Cdc12 quantified by malachite green assays over a range of initial GTP concentrations (20–300 µM). Data points show arithmetic means ± s.d. of three independent experiments, each averaged from 1–4 technical replicates. c , Representative stopped-flow fluorescence traces of EDA-GTP-BDP-FL association and dissociation with monomeric Cdc12. Global non-linear fits are shown in neon green. Kinetic parameters ( k1 , k−1 , k2 , k−2 , KD ) are geometric means with asymmetric 95% CIs from six independent association experiments (association was measured at 1.0–4.2 µM Cdc12 (four preparations) or 1.0–3.1 µM (two preparations)) and four independent dissociation experiments. d , As in c for Cdc11 with kinetic constants from four independent experiments. e , f , Stopped-flow fluorescence traces for Cdc3 ( e ) and Shs1 ( f ) revealing no detectable binding to EDA-GTP-BDP-FL or EDA-GDP-BDP-FL. However, we cannot exclude the possibility that these subunits are incompatible with the fluorescent nucleotide analogs.

Article Snippet: For association kinetics, constructs #1 and #2 were measured under pseudo-first-order conditions at final protein concentrations between 1.0 and 4.2 μM in the presence of 0.08 μM EDA-GTP-BDP-FL (Jena Bioscience), whereas construct #4 was assayed at final protein concentrations of 21 to 104 μM with 0.8 μM EDA-GTP-BDP-FL.

Techniques: Purification, Incubation, Stable Transfection, Control, Comparison, Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: A nucleotide-triggered molecular switch orchestrating septin polymerization

doi: 10.64898/2026.05.07.723466

Figure Lengend Snippet: a , Nucleotide-content analysis of purified, heat-denatured monomeric G-domains after incubation with 3 mM GTP and GDP followed by desalting. No stably bound nucleotide was detected. A protein-free desalting control is shown for comparison. b , GTP hydrolysis by monomeric Cdc10 and Cdc12 quantified by malachite green assays over a range of initial GTP concentrations (20–300 µM). Data points show arithmetic means ± s.d. of three independent experiments, each averaged from 1–4 technical replicates. c , Representative stopped-flow fluorescence traces of EDA-GTP-BDP-FL association and dissociation with monomeric Cdc12. Global non-linear fits are shown in neon green. Kinetic parameters ( k1 , k−1 , k2 , k−2 , KD ) are geometric means with asymmetric 95% CIs from six independent association experiments (association was measured at 1.0–4.2 µM Cdc12 (four preparations) or 1.0–3.1 µM (two preparations)) and four independent dissociation experiments. d , As in c for Cdc11 with kinetic constants from four independent experiments. e , f , Stopped-flow fluorescence traces for Cdc3 ( e ) and Shs1 ( f ) revealing no detectable binding to EDA-GTP-BDP-FL or EDA-GDP-BDP-FL. However, we cannot exclude the possibility that these subunits are incompatible with the fluorescent nucleotide analogs.

Article Snippet: Constructs #3 and #5 were measured at final concentrations of 104 μM protein with 2.5 μM EDA-GTP-BDP-FL or EDA-GDP-BDP-FL (Jena Bioscience).

Techniques: Purification, Incubation, Stable Transfection, Control, Comparison, Fluorescence, Binding 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: (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