Fig. 3: Pi-mediated competitive inhibition of Alexa647-ATP binding to HMM with nonfluorescent ADP (D) locked to the active site by the presence of vanadate (V_i).

a Time-averaged Alexa647-ATP binding reflected in fluorescence projections (Zprojection standard deviation in ImageJ) of 15 min videos (50 ms exposure time/frame) under 0 and 43 mM added [Pi] after preincubation with HMM (34.3 pM) with or without pretreatment with ATP and vanadate (HMM:ATP:vanadate; 1:117:583 molar ratios). In control experiments, the coverslips were coated only with bovine serum albumin (BSA). Note a decrease in the total number of Alexa647-ATP binding spots per image with added Pi. The observed decrease in spot number between HMM and HMM * D·Vi (at 0 mM added Pi) should be treated with caution since some of the heads could be lost during HMM * D·Vi complexes preparation for different reasons³⁵. Scale bars, 5 µm. b Hotspots number estimation under different experimental conditions. c Cumulative frequency distributions of Alexa-nucleotide dwell time events on HMM surface hotspots. Data either with or without nucleotide pocket blocked by nonfluorescent ADP (D) and vanadate (HMM * D·Vi complex). The data were best fitted (solid lines) by triple exponential functions (no added Pi) or double-exponential functions (at 43 mM added Pi). d Fractional phases from fittings in c, under denoted experimental conditions, normalized to the control distribution (HMM, without added Pi, N_dwell = 1233). Note that blocking of nucleotide pocket by ADP·vanadate only reduced myosin basal ATPase phase (green), as expected. In contrast, the addition of Pi also reduced amplitudes of unspecific Alexa647-ATP binding phases (blue, orange). Please see Supplementary Table 2 for best fit mean values ± 95% CI. e Rate constants obtained from fittings to data in c. Empty squares are from the fitting of the background (BSA). Temperature: 23 °C.