Qk system

Binding affinities between Stx1a(1–265) or Stx1a(25–265) and Munc18a or Munc18a(S306E/S313E) were measured using bio-layer interferometry in an Octet QK system equipped with Streptavidin-coated biosensor tips (ForteBio, Menlo Park, CA). Analyses were performed in 20 mm HEPES, pH 7.5, 90 mm NaCl, 20 μm EGTA, 0.1% β-mercaptoethanol, 0.05 mg/ml BSA at 30 °C using 96-well microplates (Greiner) shaking with a speed of 1,000 rpm during all experimental steps. Streptavidin biosensor tips were loaded over 10–30 s or up to a binding height of ∼1 nm in Stx1a(1–265) or Stx1a(25–265) containing a C-terminal Avitag sequence. To monitor complex formation between Munc18a and the syntaxin-1A constructs, the syntaxin-1A-loaded tips were dipped into a Munc18a dilution series (0, 5, 10, 20, 40, 80, 160, and 320 nm for WT Munc18a and Munc18a(S306E/S313E)) and allowed to associate for 200–1,000 s. Dissociation (200–1,000 s) was performed in buffer (20 mm HEPES, pH 7.5, 90 mm NaCl, 20 μm EGTA, 0.1% β-mercaptoethanol, 0.05 mg/ml BSA).

Real-time binding kinetics between 5-Helix and biotinylated SARS-CoV-2 HR2P was recorded on an Octet QK system (Fortebio, San Francisco, CA, USA) at 25 °C. Biotinylated SARS-CoV-2 HR2P was loaded onto a streptavidin biosensor. Serially diluted 5-Helix was associated with HR2P for 1200 s, followed by another 1200 s dissociation time in PBS containing 0.02% Tween. The equilibrium dissociation constant (K_D) was calculated by Octet QK software by a 1:1 binding model [16 (link)].