4-(2-aminoethyl)benzenesulfonylfluoride

The cell culture pellets were re-suspended with 50 ml lysis buffer (100 mM Tris-HCl [pH 8], 20 mM, Imidazole, 500 mM NaCl, 1 mM TCEP-HCl (Tris(2-carboxyethyl)phosphine hydrochloride), 2% (V/V) Glycerol), supplemented with 1 ml lysozyme (50 mg/ml), 50 μl DNase I (5 mg/ml) and one tablet of protease inhibitor. Bacterial cells were lysed with a microfluidizer or French Press at ~ 20,000 psi. Lysis was considered complete when the cloudy cell suspension becomes translucent. The lysate was centrifuged for 30 min at 16,000 rpm at 4 °C. Soluble protein (supernatant) was removed into a fresh 50 ml centrifuge tube. The supernatant was then filtered through a 0.22 μm filter and kept on ice. Affinity chromatography purification was performed using a HisTrap™ FF column (5 ml) in the ÄKTA protein purification system. The column was washed with Wash buffer 1 (100 mM Tris-HCl [pH 8], 20 mM Imidazole, 2 M NaCl, 2% Glycerol, 1 mM TCEP-HCl, 0.1. mM AEBSF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride)) to remove nonspecifically bound DNA. Then the column was washed using Wash buffer 2 (100 mM Tris-HCl [pH 8], 20 mM Imidazole, 50 mM NaCl, 2% Glycerol, 1 mM TCEP-HCl, 0.1 mM AEBSF). Elution was carried out with Elution buffer 1 (100 mM Tris-HCl [pH 8], 500 mM Imidazole, 500 mM NaCl, 2% Glycerol, 1 mM TCEP-HCl, 0.1 mM AEBSF) using a linear gradient with a set target concentration of Elution buffer 1 of 50%. Protein-containing fractions were run on a 12% polyacrylamide gel. Visualization of protein bands was achieved by incubating the gel with InstantBlue stain for 5–10 min and the protein-containing fractions pooled. The protein sample was stored at 4 °C.

Serum (stored at −80°C in 100 µL aliquots), was thawed in a 25°C water bath for 10 minutes, then stored on ice prior to sample dilution. Samples were mixed by gentle vortexing for 8 seconds. A 6% serum sample solution was prepared by dilution into 0.94× SB17 supplemented with 0.6 mM MgCl₂, 1 mM trisodium EGTA, 0.8 mM AEBSF, and 2 µM Z-Block. A portion of the 6% serum stock solution was diluted 10-fold in SB17 to create a 0.6% serum stock. 6% and 0.6% stocks are used to detect high- and low-abundance analytes, respectively.

Plasmid pET-28b containing the open reading frame for YdaT from E. coli O157:H7 (UniProt ID A0A6M7H0F8) with an N-terminal His tag (GSSHHHHHHSSG) was transformed into competent E. coli BL21 (DE3) cells (Table 1 ▸). Transformed cells were plated on agar plates supplemented with kanamycin (25 µg ml⁻¹) and incubated at 37°C overnight. LB medium supplemented with kanamycin (25 µg ml⁻¹) was inoculated with one colony and left to incubate overnight at 37°C while shaking at 130 rev min⁻¹. 5 ml of the overnight culture was added to 500 ml LB medium (supplemented with 25 µg ml⁻¹ kanamycin) in 2 l flasks and incubated at 37°C with shaking at 130 rev min⁻¹. When the OD₆₀₀ reached 0.6–0.8, protein expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Upon induction, the cultures were incubated at 37°C for 4 h, centrifuged at 5000 rev min⁻¹ for 15 min, resuspended in lysis buffer [20 mM Tris–HCl, 500 mM NaCl, 20 mM MgCl₂ pH 8.0, 0.1 mg ml⁻¹ 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)] and stored at −80°C. To purify the protein, the frozen cells were thawed and DNase I was added (50 µg ml⁻¹). The cells were lysed by sonication (5 min, 5 s on and 5 s off, 70% amplitude) and the lysate was centrifuged at 18 000 rev min⁻¹ for 45 min. The supernatant was filtered (0.45 µm HAWP filter) and loaded onto a pre-packed HisTrap HP Ni²⁺-Sepharose column (GE Healthcare) pre-equilibrated in 20 mM Tris–HCl, 500 mM NaCl, 5 mM imidazole pH 8.0. The column was then washed with the same buffer until baseline stabilization. A linear gradient (0.0–1.0 M imidazole in 20 column volumes) elution was applied in 20 mM Tris–HCl, 500 mM NaCl, 1 M imidazole pH 8.0. Fractions containing the protein were concentrated and loaded onto a Superdex 200 16/90 size-exclusion chromatography (SEC) column (GE Healthcare) pre-equilibrated in 20 mM Tris–HCl, 500 mM NaCl pH 8.0. The purity of the protein was assessed by SDS–PAGE.
A truncated variant of YdaT lacking the 45 C-terminal residues (YdaT^1–96) was created by replacing Ser97 with a stop codon (TAA) via PCR amplification of the whole plasmid using primers 1 and 2 (Table 1 ▸, Supplementary Table S1) and Q5 High-Fidelity 2× Master Mix (NEB). The unmodified plasmid was degraded by incubation with DpnI for 1 h at 37°C. The mutations were confirmed by sequencing and CaCl₂-competent E. coli BL21 Star (DE3) cells were transformed with the mutated plasmids. Both proteins were expressed and purified as described for wild-type YdaT except for the use of a Superdex 75 16/60 SEC column (GE Healthcare) for the SEC purification step.

The NavAb cDNA carrying N49K mutation was kindly provided by Dr Katsumasa Irie of Nagoya University, Japan (28 (link)). The cDNA-encoding NavAb channel carrying N49K/T206A mutation was subcloned into the pET28 vector to express monomeric NavAb channels. The NavAb tandem tetramer was constructed by linking four NavAb cDNAs (N49K) with flexible linkers containing 2xGGGS and ‘LVPRGS’ thrombin-cutting sites between each subunit. The NavAb proteins were expressed in E. coli KRX host cells, and cell cultures were induced when A600 reached 0.6, by 0.1 mM isopropyl β-D-1-thiogalactopyranoside and 0.1% L-rhamnose (w/v) overnight under 25 °C. The cells were harvested and resuspended into the resuspension buffer containing 20 mM Tris–HCl, 150 mM NaCl, proteinase inhibitor cocktail (2 mM phenylmethylsulfonyl fluoride, 1 mM AEBSF, 1.5 μM Pepstatin, 1.4 μM E−64, 4 μM Bestatin), DNase I, pH 8.0, then homogenized by a Microfluidizer (Microfluidics Inc). The membranes were collected by ultracentrifugation under 4 °C, 100,000g for 3 h, then resuspended into the resuspension buffer with an additional 20 mM imidazole. The NavAb proteins were extracted from membranes by 1% (w/v) lauryl maltose neopentyl glycol (LMNG), under 4 °C for 3 h and then purified by Talon cobalt resin (Takara Inc) (23 , 28 (link)). The affinity-purified NavAb proteins were further separated by a Superdex 200 size-exclusion column using the gel filtration buffer containing 20 mM Tris, 150 mM NaCl, 0.1% LMNG, pH 8.0. The tetramer fractions of NavAb proteins were pooled and concentrated by Amicon ultrafilters. The NavAb T36C/Q115C mutant proteins for smFRET studies were bound to Talon cobalt resin and then changed into the fluorophore-labeling buffer containing 20 mM Tris, 150 mM NaCl, 0.1% LMNG, pH 7.0. The NavAb proteins bound to cobalt resin were incubated with 100 μM Cy3/Cy5 c5 maleimide (1:1) with improved photostabilities (29 ) at 4 °C for 2 h. The free fluorophores were removed by the gel filtration buffer containing 20 mM imidazole and then eluted by the gel filtration buffer containing 500 mM imidazole. The fluorophore-labeled NavAb tetramer proteins were further separated by a Superdex 200 column and then either reconstituted immediately or stored in a −80 °C freezer. The human voltage-gated proton channel hHv1 was expressed, purified, labeled, and reconstituted as described previously (23 , 24 ).

For immunoblots, cells were harvested in modified RIPA buffer (50 mM tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal CA-630 [v/v], 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1 mM AEBSF, protease inhibitor cocktail) and incubated on ice for 30 min prior to SDS–PAGE and transfer to nitrocellulose. The following antibodies were used in immunoblots: β-actin (clone AC-74, Sigma-Aldrich; dilution 1:2000), E2F1 (3742S, Cell Signaling Technology, dilution 1:1000), symmetric di-methyl arginine (SDMe) (13222S, Cell Signaling Technology, dilution 1:1000), FLAG (clone M2, F1804, Sigma, 1: 1000), GAPDH (clone 6C5, MAB374, Millipore, 1:2000). Uncropped versions of immunoblots are presented in the supplementary figure 11.