Pa25031

NusA was labeled with Cy5-maleimide dye (PA25031, GE Healthcare) using an 8:1 molar ratio of dye to protein in a total volume of 200 μL (~ 20 μM protein) of labeling buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5 mM TCEP, 0.1 mM Na₂EDTA). After ~ 4.5 hours incubation at 4°C, the reaction was quenched upon addition of excess 2-mercaptoethanol. NusA-Cy5 and the free dye were separated by ion exchange chromatography. The labeling reaction was loaded on Mono Q column and washed with excess amount (over 20 column volume) of ion exchange buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5 mM TCEP, 0.1 mM Na2EDTA) to remove the free dye. Then NusA-Cy5 was eluted from the column using a NaCl gradient from 0.2 M to 2 M. The fractions containing NusA protein were confirmed by SDS-PAGE, the purified labeled protein was mixed in 1:1 ratio with storage buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5 mM TCEP, 0.1 mM Na₂EDTA, 50% glycerol) and flash frozen for storage at −80°C. Labeling stoichiometry was determined as ~ 0.6 for Cy5/NusA using the absorbance measurements at A₂₈₀ and A₆₅₀.

H1.0 G101C K195C, in which cysteines were located at either end of the H1 CTD, was incubated in 50 mM DTT for 1 h on ice to reduce the cysteines, then DTT was removed by Bio-Rex chromatography, and fractions were immediately frozen on dry ice. Fractions containing reduced H1.0 G101C K195C were treated with 5∼10-fold excess of either maleimide-Cy3, or maleimide-Cy5, or a 50/50 mix of both (GE Healthcare; catalog nos.: PA23031 and PA25031) for 30 min at room temperature in the dark. Free dyes were removed by another round of Bio-Rex cation-exchange chromatography. Concentration of fluorophore-labeled H1.0 was determined by quantitative comparison with an H1 standard, as described previously.

The purified dcapsid proteins were labelled with the Cy5 fluorophore based on the chemical reaction between the ¹⁰⁸cysteine of capsid and Cy5-maleimide (PA25031, GE Healthcare Life Sciences). The labelled molecules were separated from the unreacted and excess dyes with a PD Mini-Trap G-25 desalting column, and mixed at 10–50 nM concentration with 2 fold serially dilutions of EGCG, EC or heparin in reaction buffer (150 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 0.05% Tween-20, pH 7.4). The binding reactions were fixed into equal volume and incubated at room temperature for 15 min. The mixtures were then enclosed in standard pretreated glass capillaries and analyzed with MST machine (Monolith NT.115, NanoTemper Technologies, Munich, Bavaria, Germany), and the Kd values of each reaction were computed by NanoTemper Analysis software (Version 2.1, NanoTemper Technologies, Munich, Bavaria, Germany).