Fluorescence Polarization Assay for HalA2LP

FP experiments were conducted as previously described^{58 (link)} with the following changes: HalM2 was serially diluted in GF buffer, then added to FP buffer (50 mM HEPES pH 7.5, 30 mM KCl, 5% glycerol, 1 mM MgCl₂, 1 mM TCEP, 0.25 mM AMP-PNP, 0.0025% IGEPAL-CA630, 10 nM Fluor-HalA2LP). The sample was allowed to incubate at RT for 15 min, then the parallel and perpendicular fluorescence intensities were measured. All experiments were conducted in triplicate in 386-well solid black polystyrene microplates (Corning) on a Synergy H4 Hybrid plate reader (BioTek). Data analysis was performed using Origin 9.6. Polarization was calculated using the following formula: P = (I_|| - I_⊥) / (I_|| + I_⊥). Competition FP assays were conducted using serially diluted HalA2LP analogs in GF buffer. Each dilution was mixed with FP Buffer + HalM2 (14 μM). Curves were fit to either non-linear dose-response or hyperbolic function. K_i was calculated using the following equation: K_i = IC₅₀ / (1 + [L]/K_d) where [L] = 10 nM Fluor-HalA2LP.