Thermal Shift Assays

Thermal shift experiments were based on a previously established protocol.³⁸ Assays were performed in a Hard-Shell 96-well PCR plate (Bio-Rad, Hercules, CA). Solutions were prepared by mixing 2 μL of ×10 SYPRO Orange protein gel stain (Thermo Fisher Scientific, Waltham, MA) and 1 mg/mL BauF in 100 mM potassium phosphate, pH 7.5, to a total volume of 20 μL. For ligand-binding studies, the protein was incubated for 10 min with 0.01–1 mM Acb-Fe, 0.01–0.8 mM ox-pre-Acb-Fe, 1 mM NAD(P)⁺, or 10 mM DT before analysis. The plate was sealed with the MicroAmp optical adhesive film (Thermo Fisher Scientific) and analyzed using a CFX qPCR (Bio-Rad) programmed to heat from 20 to 90 °C at a rate of 2 °C/min. Changes in the fluorescence were measured every 30 s with a λ_ex 450–490 nm and λ_em 610–650 nm. The fluorescence emissions were analyzed to determine the enzyme melting temperature (T_m) using the Boltzmann sigmoidal curve with Graphpad Prism (Huynh, 2015) (eq 3).

where y is the fluorescent emission, x is the temperature, A₁ is the initial fluorescence, A₂ is the maximal fluorescence after enzyme melting, A₃ is the steepness of the curve, and T_m is the temperature of protein melting. The T_m values were plotted against the concentration of ligand and the K_D was determined using eq 4.³⁹

where y is the T_m, L is the T_m when no ligand is present, H is the T_m at saturating concentration of ligand, P is the protein concentration in the same units as ligand concentration, K_D is the dissociation constant, and x is the ligand concentration.