Fig. 2: Single molecule ATPase.
a Two principles for surface immobilization of myosin motor fragments. b Time-averaged Alexa-ATP fluorescence projections of 15 min videos (50 ms exposure time/frame). (i) Simple HMM deposition at 34.3 pM (left) and 343 pM (right). (ii) Optimized HMM deposition following high [ATP] wash. Only spots defined by previous position of F-actin filament were included in analysis. (iii) Image of optimized actomyosin deposition followed by low [ATP] wash showing co-localization of Alexa-ATP fluorescence (gray spots) and F-actin filaments (yellow). Only spots co-localizing with F-actin filaments were included in analysis. Bars, 5 µm. c Representative time traces of (i) single-molecule Alexa-Ph bleaching, (ii) HMM basal ATPase, and iii, actin-activated HMM ATPase. Bleaching was observed either using standard in vitro motility assay (IVMA) buffer or optimized TIRF microscopy buffer (TIRFM) as described in the text (for details see Supplementary Information sections 1.2 and 1.9), whereas ATPase traces were from experiments using TIRFM buffer. d Cumulative frequency distributions of single-molecule Alexa-Ph until bleach or first blinking event. The distributions are fitted by single exponential functions (solid lines). e Cumulative frequency distribution of Alexa-nucleotide dwell time events on HMM surface hotspots (IVMA buffer—simple deposition of HMM, TIRFM buffer-optimized deposition of HMM) were fitted with double-exponential functions (solid lines). IVMA data from 118 HMM molecules, Ndwell = 2112, TIRFM data from 45 HMM molecules, Ndwell = 785. f Amplitudes and rate constants from the fitting of data in e. Note appreciably lower rate constant values in TIRFM compared to IVMA buffer. g Cumulative frequency distributions of Alexa-nucleotide dwell time events comparing HMM basal (re-plotted from panel e) and actin-activated ATPase activity. Actin-activated ATPase data were obtained from 37 actomyosin hotspots, Ndwell = 879. The actin–myosin data were fitted with triple exponential functions, whereas basal ATPase data were fitted by double-exponential function as in f. h Rate constants obtained from fittings to data in f. i Amplitudes from fitting the data in f. Error estimates refer to 95% confidence intervals derived in the regression analysis. Temperature: 23 °C.
