The NaK2K construct (NaK D66Y and N68D double mutant; plasmid provided by Y. Jiang) is missing the first 19 amino acids and has a C-terminal hexahistidine tag. The NaK2K construct was cloned from the original pQE60 vector into a pET28a expression vector using the NCO I and Hind III cleavage sites on both plasmids (protein sequence: MAKDKEFQVLFVLTILTLISGTIFYSTVEGLRPIDALYFSVVTLTTVGDGNFSPQTDFGKIFTILYIFIGIGLVFGFIHKLAVNVQLPSILSNLVPRGSRSHHHHHH).

The protein was expressed in E. coli OverExpress C43(DE3) (Lucigen) using 2 liters of deuterated M9 medium. The cells were adapted to D2O conditions as described previously (24). Deuterated M9 medium (D2O 99.9% D; D214, Eurisotop) was supplemented with α-ketobutyric acid (60 mg/liter; CDLM-7318-PK, Cambridge Isotope Laboratories), d-glucose 13C (4 g/liter; 552151, Sigma-Aldrich), and lyophilized deuterated 15N ammonium chloride (1 g/liter; 299251, Sigma-Aldrich). At an OD600 (optical density at 600 nm) of 0.8, the cultures were induced with 0.4 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) and grown for 24 hours at 25°C. Additional labeled deuterated glucose (1 g/liter) and deuterated 15NHCl (0.25 g/liter) were added to the cultures 6 hours after induction. Cells were harvested and resuspended with 10 ml of lysis buffer [50 mM tris (pH 7.5), 100 mM NaCl, 1 mM MgCl, and protease inhibitor cocktail (11836170001, Sigma-Aldrich)] per gram of cell pellet. The sample was lysed using a microfluidizer (LM10, Microfluidics) with five cycles of a working pressure of 15,000 psi. The membrane fraction was isolated by ultracentrifugation at 150,000 relative centrifugal force (rcf) for 2 hours and solubilized in 80 ml of solubilization buffer [50 mM tris (pH 7.5), 100 mM NaCl, and 40 mM DM (n-Decyl-β-maltoside; GLYCON Biochemicals)] at room temperature for 3 hours. The protein was batch-purified by immobilized metal affinity chromatography using 5 ml of bed volume of TALON Superflow beads (GE Healthcare Life Sciences). Protein concentrations were determined using Bradford reagent supplemented with α-cyclodextrin (5 mg/ml; AppliChem), and 5.4 mg of protein was mixed with E. coli total lipid extract (100500, Avanti Polar Lipids) in a ratio of 2:1 (w/w). H/D back exchange and proteoliposome formation were performed by dialysis for 8 days at 100× dilution against 500 ml of 100% H2O with 20 mM tris (pH 8.0) and 50 mM KCl, followed by ultracentrifugation at 300,000 rcf for 4 hours at 4°C. Half of the pellet was removed for NMR experiments (sample A, K+). The second half of the sample was resuspended in 1 ml of 20 mM tris (pH 8.0) and kept rotating at 4°C. The buffer was refreshed twice over the course of 14 days, each time after ultracentrifugation at 150,000 rcf for 30 min at 4°C.

From this, half of the sample was removed for NMR experiments (sample B, no ions). The other half of the sample was resuspended in 1 ml of 20 mM tris (pH 8.0) with 50 mM KCl. The sample was kept rotating at 4°C for 8 days, during which time the buffer was refreshed once and used for NMR experiments (sample C, washed to K+).

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