Single-molecule FRET experiments were performed on surface-immobilized molecules employing a prism-based total internal reflection microscope (Zeiss). Labeled molecules were immobilized inside a coverslide chamber (a pair of quartz and glass slides assembled together by Parafilm stripes) using a BSA-biotin and streptavidin anchoring. Stock solutions of the labeled sample with a concentration of ∼5 pM were used for immobilization. The excess of non-immobilized molecules was washed out by flushing the chamber with dilution buffer (20 mM Tris–HCl buffer (pH 7.5), with 25 mM LiCl or KCl). Detailed protocols of these experimental procedures are published elsewhere (32). Prior to imaging, the coverslide chamber was flushed with an imaging buffer consisting of the dilution buffer supplemented with an oxygen scavenging system composed of 2 mM Trolox (Sigma Aldrich), glucose oxidase (Sigma Aldrich, 17 U/ml), catalase (Sigma Aldrich, 260 U/ml) and glucose (Sigma Aldrich, 4.5 mg/ml). Fresh imaging buffer was flushed into the chamber every 20 min, to avoid pH gradients. The excitation of fluorophores was achieved using an alternating laser excitation scheme (33) with 514 nm Ar-ion and 630 nm dye lasers. Fluorescence from the donor and acceptor fluorophores was spatially separated onto the EMCCD camera (Andor, iXON 3) by a wedge mirror. Movies were recorded with a 200 ms integration time per frame with a total length of 600 s (10 min) and were analyzed using the home-built iSMS software (34). Only the trajectories with single-step donor and/or acceptor photobleaching, or showing clear anti-correlated donor–acceptor dynamics, were selected for further analysis. The FRET efficiencies were obtained from the donor and acceptor fluorescence intensities as: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}\begin{equation*}E = \frac{{{F_A}}}{{{F_A} + \gamma {F_D}}}\end{equation*}\end{document}