Standard capillaries

The purified AK concentration was adjusted to 2–20 μM with labeled buffer. The solid fluorescent dye (NT-647-NHS) was appended with 100% DMSO (30 μL) and dissolved thoroughly after vortex vibration. Then the dyes were adjusted to 2–3 folds of the protein concentration with the marker buffer. Protein and the solid fluorescent dye were mingled in equal proportion for dark reaction 30 min, and then purified according to the purification kit operation. The unlabeled substrate Asp and inhibitor Lys were diluted in the enzymatic activity measurement system, and the final concentration ranged from 0.5 M to 15 M and from 0.1 M to 3 M, respectively, which they were incubated with the labeled protein (40 nM) at room temperature for 10 min. Then the samples were loaded into the Standard Capillaries from Nano Temper Technologies. Thermophoresis was measured 30 min after incubation at 28 °C on Monolith NT.115.^{22 (link)} The dissociation constant (K_d) values were fitted by means of the MO. Affinity analysis software.

According to previously classic method, the binding was calculated for MST Monolith NT. 115 (Nano Temper Technologies, Germany) [38 (link), 39 (link)]. Using a NT-647 dye (Nano Temper Technologies, Germany) incubating 0.5 µM of purified recombinant proteins with A range of ligands from 0 to 5 µM were for 5 min, and using the final concentration of 20 nM to the thermophoresis experiment. The selected compounds in DMSO were made a 16 point dilution series. Each compound dilution series was subsequently transferred to protein solutions (10 mM Tris–HCl and 100 mM sodium chloride pH 7.5, 0.05% Tween-20). The labeled TMV CP with each dilution point (1:1 mix) were incubation 15 min at room temperature, then the standard capillaries (NanoTemper Technologies, Germany) were filling into the samples. Under a setting of 20% LED and 40% IR laser, using the Monolith NT.115 microscale thermophoresis system (NanoTemper Technologies, Germany) to measure. Laser on time was set at 30 s, and laser-off time was set at 5 s. Using the mass action equation in the Nano Temper software, the Kd values were calculated from the duplicate reads, each experiments average of three replicates.

P5PA was fluorescently labeled using RED-NHS Second Generation Protein Labeling Kit (NanoTemper). Briefly, 90 μl of purified apo-P5PA at 10 μM was mixed with 10 μl of the provided labeling dye at 300 μM. The mixture was incubated in dark for 30 min and loaded to a special gravity column that is pre-equilibrated in PBS with 0.05% Tween-20 (PBST). After washing the column with 550 μl of PBST, another 450 μl of PBST was added to elute the fluorescently labeled apo-P5PA. The yield of the labeled P5PA was determined by 280-nm readings made on a Nanodrop.
A target compound was successively diluted in PBST by a dilution factor of two to make a total of 16 different concentrations. Then, 10 μl of the labeled P5PA was added to an equal volume of the diluted compounds and loaded to standard capillaries (NanoTemper). The Binding Affinity Mode of Monolith NT.115 (NanoTemper) was used to detect fluorescence changes and deduct K_d. The corresponding final concentrations of the labeled P5PA and the tested compounds were listed in Table S2.