Protein preparation

OmpLA was prepared and purified as described elsewhere (30, 31). Briefly, tag-free OmpLA without signal sequence was produced as inclusion bodies (IBs) in Escherichia coli BL21(DE3) cells. IBs were isolated, sonicated, and solubilized in 8 M urea. Unfolded OmpLA was separated from residual impurities by centrifugation for 30 min at 7000 × g and 4°C, refolded by drop dilution in buffer (20 mM Tris and 2 mM EDTA (pH 8.3)) containing 10 mM lauryldimethylamine N-oxide (LDAO; Sigma-Aldrich, St. Louis, MO), and folded protein was further isolated with anion exchange chromatography. For single-molecule fluorescence experiments a protein variant was engineered with two Cys residues at positions 125 and 234 (Fig. S1). After protein production and purification as described above under reducing conditions (5 mM tris(2-carboxyethyl)phosphine), the protein was site-specifically labeled via thiol-maleimide chemistry with maleimide-functionalized donor (ATTO532; Atto-Tec, Siegen, Germany) and acceptor (ATTO647N; Atto-Tec) fluorophores. The labeled protein was separated from unbound dyes by size-exclusion chromatography. The melting point of labeled (T_m = (90.2 ± 0.2)°C) and unlabeled (T_m = (91.6 ± 0.6)°C) OmpLA agreed well in circular dichroism (CD) spectroscopy measurements, indicating only little effect of the labels on the protein stability.