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