Protein expression, site-specific labeling, and purification

C-terminally His₆-tagged WT KcsA and most mutants were expressed from a pQE60 vector (QIAGEN) in either BL21 (DE3) T1-R (Sigma-Aldrich) or XL-1 Blue (Agilent Technologies) competent cells and grown in Luria broth (LB) with ampicillin. Cells were transformed with the appropriate vectors, and the resulting plates were scraped and pooled for inoculation the following day.

The site-specific labeling protocol was adapted from Marley et al. (2001). Cells were grown with aeration at 37°C until they reached an OD₆₀₀ of ∼0.9 when they were spun at 5,000 rpm for 10 min at 4°C. The supernatant was removed and the cell pellet was washed with M9 salts (Sigma-Aldrich). Each cell pellet from 1 liter of media was then resuspended into 250 ml of minimal media (50 ml of 5× M9 salts, 100 µM CaCl₂, 1 mM MgSO₄, 0.25 g AmCl₂, 1 g dextrose, 2.5 ml of 100× BME vitamins (Sigma-Aldrich), and 100 µg/ml ampicillin) supplied with 20 mg [¹⁵N]His or [¹³C,¹⁵N]His (uniformly labeled, Cambridge Isotope Laboratories). The remaining unlabeled amino acids were added in excess. The cells were recovered by aeration for 1 h at 37°C if induction occurred at 37°C or by aeration for 40 min at 37°C followed by 20 min of aeration at the intended induction temperature. After recovery, cells were induced by the addition of 0.5 mM IPTG. All KcsA mutants and WT were induced at 37°C for 4 h except for H25R (30°C, 4 h). After induction, cells were collected by centrifugation at 5,000 rpm for 10 min at 4°C. Cell pellets were stored on ice overnight at 4°C.

Purification of KcsA was adapted from Thompson et al. (2008), with minor modifications. Cell pellets were resuspended in buffer containing 50 mM Tris, pH 7.6, and 100 mM KCl and lysed by sonication. Membrane extraction occurred at room temperature for 2 h with 50 mM n-decyl maltoside (DM). Detergent-solubilized extract was separated by centrifugation for 45 min at 17,500 rpm at 4°C. Crude lysate was applied to a Hi-Trap chelating column charged with Ni⁺² resin and washed with buffer B (100 mM KCl, 20 mM Tris, and 5 mM DM, pH 7.5) and 30 mM imidazole. Protein was eluted with buffer B and 300 mM imidazole and concentrated before incubation with chymotrypsin (enzyme to protein ratio was 1:50, by weight) for 2–3 h at room temperature. The proteolyzed protein was applied to a Sephadex-200 FPLC column equilibrated with buffer B, and protein eluting at the correct retention volume for KcsA ΔC was collected and pooled. Protein concentration was assayed by UV absorption spectroscopy at 280 nm and was calculated using an extinction coefficient of 33,570 M⁻¹cm⁻¹.