Cy5 maleimide dye

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dielaidoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) and E. coli polar lipid extract were purchased from Avanti polar lipids. Tris was purchased from Promega. Potassium chloride and imidazole were purchased from Merck. Silicon wafers were from universitywafers.com. RTV 615 PDMS was purchased from Momentive. Nitric acid was from Merck. Cy3-NHS and Cy5-maleimide dyes were purchased from GE Healthcare. Phosphoenolpyruvic acid (PEP) was from Alfa Aesar. All other materials were from Sigma-Aldrich unless otherwise stated.

Corresponding MxA^MO2 derivatives were labelled with Cy3- and Cy5-maleimide dyes56 (link) (GE Healthcare), as donor and acceptor respectively. For labelling, the protein was incubated with a fivefold molar excess of respective dye for 1 h at 4 °C in the buffer containing 20 mM HEPES (pH 7.0), 150 mM NaCl, 4 mM MgCl₂ and 0.5 mM TCEP. The reaction was quenched by the addition of L-cysteine with 100-fold molar excess of two dyes and then the excess free dyes were removed using Zeba spin desalting columns (Thermo Scientific). In addition, for imaging experiments on dimeric GDP·AlF₄⁻-bound proteins, the samples were prepared as described in Supplementary Fig. 5. Dye-labelled protein without His₆-tag was incubated with His₆-tagged protein which was not labelled (yellow) in the presence of 1 mM GDP, 1 mM AlCl₃ and 10 mM NaF. These proteins were subsequently applied to SEC and the fractions of the dimers were collected for immobilization.

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.