SthK channel was purified as described in Marchesi et al., 2018 (link). bR and ChR1 proteins were bought from Cube-Biotech. The purified protein or the different mixtures (as indicated in the Results section) were brought at a total concentration of 0.5 mg/ml in a buffer containing 20 mM HEPES, pH 7.8, 150 mM KCl, and 0.1% N-dodecyl β-D maltoside (DDM), and aliquoted into 100 µl samples. A lipid mixture of 1,2-dioleolyl-sn-glycero-3-phosphocholine, 1,2-dioleolyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleolyl-sn-glycero-3-phospho-L-serine at 8:1:1 ratio (Avanti Polar Lipids) was added at a lipid-to-protein ratio of 1 (wt/wt). The different ternary mixtures of protein-lipid-detergent were sonicated in an ice-bath sonicator for 2 min and subsequently equilibrated on an orbital shaker (200 rmp) for 2 hr. Detergent was removed by hydrophobic adsorption adding twice ~5 mg of wet bio-beads SM-2 and equilibrating under gentle shaking (200 rmp) for 1 hr at RT and overnight at 4°C, respectively. Before use samples were diluted 1:3 in adsorption buffer (20 mM HEPES, pH 7.8, 150 mM KCl).
The reconstituted membrane proteins were adsorbed on freshly cleaved mica for 30 min in a humid chamber. The samples were gently rinsed with imaging buffer (150 mM KCl, 10 mM HEPES, pH = 7.8) and subsequently used for AFM imaging and unfolding.
The reconstituted membrane proteins were adsorbed on freshly cleaved mica for 30 min in a humid chamber. The samples were gently rinsed with imaging buffer (150 mM KCl, 10 mM HEPES, pH = 7.8) and subsequently used for AFM imaging and unfolding.
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