Fig. 1: Single-molecule stoichiometry measurements of DmpR oligomers.
From: Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR
a Domain organisation of the eGFP-DmpR protein used for single-molecule photobleaching (SMPB). b Schematic overview of the experimental design of SMPB assays. c A representative EMCCD image including four major species of eGFP-DmpR proteins (monomer, dimer, trimer and tetramer). Asterisks represent the signal from presumable protein aggregates. All data are representative of five replicates with similar results. Scale bars, 5 μm. d Representative time trajectories of the eGFP emission signals. The stoichiometry of the eGFP-DmpR proteins was determined by counting the number of eGFP photobleaching steps. Light blue lines are eGFP emission traces. Pink lines represent stepwise fits of the traces. e A representative time trajectory of the signal from presumable protein aggregates. f–j Distribution of photobleaching steps of eGFP-DmpR. The pie graphs above the histograms depict the ratio of dimers and tetramers for each condition. Events with more than eight photobleaching steps were categorised as aggregates (A). Data are presented as mean ± SD from three independent experimental replicates with n ≥ 180 individual molecule (Counts).
