Fig. 2: Single-molecule Alexa647-ATP binding to HMM with increasing [Pi].
From: Multistep orthophosphate release tunes actomyosin energy transduction
a Time-averaged Alexa-ATP binding event is represented as fluorescence projections (standard deviation function in Fiji [ImageJ]) of 15 min videos (50 ms exposure time/frame; Movie S1) under increasing added [Pi] (mM). HMM incubation at 34.3 pM. In control experiments, the coverslips were coated only with bovine serum albumin (BSA). Scale bar, 5 µm. b Hotspot numbers decrease by increasing added [Pi]. The experiments were performed on two different occasions (series 1, black; series 2, light blue). The numbers (N, point estimates in counting process) were normalized to the number of hotspots (series 1 = 51 and series 2 = 26) in control experiments (open square) without added [Pi], but with estimated background [Pi] ≤0.06 mM. Error bars represent standard deviation (SD) in a Poisson process, estimated as √N. The curved line added to guide the eye represents a fit to the sum of two Hills equations39 with Kd1 ~0.3 mM and Kd2 ~16 mM. Experiment with HMM and control experiment (BSA) at 43 mM added [Pi] yielded the same number of hotspots (1). Outlier indicated by dashed circle was not included in the fitting. c Cumulative frequency distribution of Alexa-nucleotide dwell time events (series 1) on HMM surface hotspots under increasing added [Pi] were best fitted (solid lines) with triple exponential (0–5.4 mM), double exponential (10.4 and 22.5 mM) or single exponential functions (43 mM). d Fractional phases from fittings in c normalized to the number of events in control distribution without added Pi (Ndwell,series1 = 745, Ndwell,series2 = 374). Note that the amplitudes of all phases were reduced by increasing [Pi]. Slow unspecific ATP binding phase (orange) is not well resolved when [Pi] > 5.4 mM and myosin basal ATPase phase (green) is not detected at 43 mM [Pi]. Best fit mean values ± 95% CI in Supplementary Table 1. e Rate constants obtained from fittings to data in c. Empty squares are from the fitting of the backgrounds (BSA). Temperature: 23 °C.
