MST assay was carried out as previously described (57 (link)). Briefly, recombinant CRP and AceE were purified with amylose resin (New England BioLabs). The purified CRP (10 μM) was incubated with 30 μM labeling dye for 30 min, followed by 16 serial twofold dilutions into buffer containing DNA. After 5 min of incubation, 4 μl of each reaction was enclosed in premium-coated glass capillaries and subjected to MST on a Monolith NT.115 NanoTemper instrument, at 40% MST power and 40% light-emitting diode power. Data were analyzed by MST software (MO.Affinity, Munich, Germany). The purified AceE and N-hydroxysuccinimide (NHS) ester fluorescent dye solutions were mixed at a ratio of 1:1 and incubated in the dark at room temperature for 30 min. Serial dilutions of ampicillin or pyruvate with PBS buffer were mixed with 20 μM NHS fluorescent-labeled AceE and then incubated at room temperature for 5 min. The sample was loaded into MST-glass capillaries,
and a NanoTemper Monolith NT.115T instrument was used for MST analysis. By plotting the concentration of antibiotic or metabolite as the per mil change of normalized fluorescence [∆Fnorm (‰) = (Antibiotic or metabolite fluorescence − Control fluorescence) / Control fluorescence], curve fitting was performed using GraphPad Prism software 8.0, and dissociation constant (Kd) values were determined. Three independent samples were carried out.
and a NanoTemper Monolith NT.115T instrument was used for MST analysis. By plotting the concentration of antibiotic or metabolite as the per mil change of normalized fluorescence [∆Fnorm (‰) = (Antibiotic or metabolite fluorescence − Control fluorescence) / Control fluorescence], curve fitting was performed using GraphPad Prism software 8.0, and dissociation constant (Kd) values were determined. Three independent samples were carried out.
