Labeling of MTSs was performed through dual cysteine labeling and subsequent purification to separate the population of sensors with a single donor and acceptor dye. The cysteines were first reduced with 2 mM tris(2-carboxyethyl)phosphine for 30 min at room temperature and buffer exchanged into labeling buffer [50 mM phosphate buffer, 150 mM NaCl, and 1 mM EDTA (pH 7.4)] using three 7K Zeba desalting columns (89883, Thermo Fisher Scientific) in series. Alexa 546 and Alexa 647 maleimide dyes were added at a protein:donor:acceptor ratio of 1:1.5:2 for 1 hour at room temperature and overnight at 4°C. To help remove free dye and exchange the protein into fast protein liquid chromatography (FPLC) buffer A [50 mM tris buffer (pH 8) and 5 mM β-mercaptoethanol], the solution was passed through two PD MiniTrap desalting columns (45001529, GE Healthcare) in series. To separate out the sensors with a single donor and single acceptor, we used an AKTA Pure FPLC (GE Healthcare) with a MonoQ PC 1.6/5 (GE Healthcare) ion exchange column and a 10 mM/ml linear salt gradient with buffer B [50 mM tris (pH 8), 5 mM β-mercaptoethanol, and 2 M NaCl]. Fractions were characterized using SDS-PAGE, UV-Vis spectroscopy, and single-molecule imaging. The desired fractions were concentrated and exchanged into PBS using 3K centrifugal filters (Amicon) and stored at −80°C.

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