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 ) SDS-PAGE analysis of bacterially expressed, purified CHIKV nsP2. 13 µg of purified nsP2 at 95% purity is shown. Broad molecular weight markers (Mr) and corresponding molecular weights are indicated. Full expression and purification of recombinant wild type nsP2 are outlined in . (B-C) AlphaFold3 models of the nsP2 ATPase active site generated without (B) or with (C) bound ATP and Mg²⁺. Walker A (K192, S193) and Walker B (D252, E253) residues are highlighted. Hydrogen-bonding (black) and metal-coordinating (green) interactions are indicated. (D) ATP hydrolysis by wild-type nsP2 monitored by thin-layer chromatography. 0.01 µM nsP2 was mixed with 1 mM ATP and conversion of ATP to ADP was monitored. The TLC plate shown is one representative wild-type experiment from the quantification in (E/F). (E) ATPase activity of nsP2 wild-type and active-site derivatives purified using Ni-NTA spin columns. Column elutions containing SUMO-nsP2 proteins were used for ATPase reactions which contained 1 µM enzyme (0.01 µM for wild type) and 1 mM ATP. Reactions were quenched at various time points and resolved via TLC. Rates were determined by linear regression of ADP formation. (F) ATPase specific activities for nsP2 wild-type and derivatives. Rates from panel (E) (slope = µM ADP·min⁻¹) were converted to specific activity (pmol ADP·min⁻¹·µg⁻¹ nsP2) using the enzyme concentration and molecular weight of SUMO-nsP2. Values are reported as mean ± SE with 95% confidence intervals.

Article Snippet: Precipitated protein was collected by centrifugation at 70,000 × g for 30 minutes, resuspended in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20% glycerol, 1 mM TCEP with 10 mM imidazole), and loaded onto Ni-NTA spin columns (QIAGEN, #31014) equilibrated in buffer A.

Techniques: SDS Page, Purification, Molecular Weight, Expressing, Recombinant, Generated, Thin Layer Chromatography, Activity Assay, Concentration Assay