Proteoliposomes were adsorbed onto freshly cleaved mica for 30 min and rinsed with SMFS buffer several times to remove nonadsorbed and weakly adsorbed proteoliposomes. Membrane patches containing reconstituted proteins were localized by AFM imaging. SMFS experiments were carried out with the same AFM (Nanowizard II Ultra; JPK Instruments AG, Berlin, Germany) having an 850-nm laser detection system. SMFS was conducted using Si3N4 cantilevers (OMCL RC800PSA; Olympus, Tokyo, Japan) having a nominal spring constant of 0.05 N·m−1 and resonance frequency of 18 kHz. Cantilevers were calibrated applying the equipartition theorem (42) before and after of each experiment. A fresh sample and a new cantilever were used in each experimental day, and >350 days were needed to conduct all SMFS experiments described in this work. The volume of buffer, electrolyte concentration, and temperature were monitored and kept constant during the experiments.

To pick up and mechanically unfold a single LacY molecule, the AFM stylus was approached to the surface of LacY proteoliposomes until reaching a contact force of ~700 pN, which was applied for 500 ms. To enhance the probability of attaching LacY from C-terminal end, polyGly extension was engineered on the C-terminal end of LacY. This 36–amino acid–long polyGly tail followed by a His8-tag increased the probability to pick up C-terminal end of LacY 10-fold (~0.1%, n = 2974) with an AFM stylus (17). Upon complete mechanical unfolding and stretching, the 461–amino acid–long LacY polypeptide (417–amino acid wild-type LacY elongated by 36–amino acid–long polyGly extension and a His8-tag) shows the force peak pattern extending to ~120 nm (17). To completely unfold and extract individual LacY transporters from the membrane, the AFM cantilever was retracted by ≥190 nm at a constant speed of 0.7 μm·s−1.

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