All of our single-molecule experiments were performed using a prism-based TIRF microscope that was equipped with a 532 nm laser and 100x NA, 1.4 oil-immersion objective (Plan Apo, Nikon), as previously described (6 (link),22 (link)). Experiments were carried out at room temperature (22°C) in a temperature-controlled laboratory that is stabilized to within ±0.5°F. Sample solutions were prepared using the standard imaging buffer unless otherwise specified. These solutions contained the oxygen scavenging and triplet quenching system used only for smFRET measurements: 165 U/ml glucose oxidase (Sigma), 0.8% (w/v) D-glucose (Sigma), 2170 U/ml Catalase (Sigma) in a Trolox solution (≥1 mM, Sigma). Protein (gp32) solutions at various concentrations containing these oxygen scavenging and triplet quenching system were incubated in a microfluidic sample chamber for 2–3 min before data acquisition. Unless otherwise specified, data were acquired without flushing the unbound proteins from the sample chamber. Data sets were generally collected on the same day for a given experiment (e.g. salt concentration dependence of a particular substrate, gp32 concentration dependence), and the results of these experiments were reproduced on separate days.