Lifetime measurements as a function of ATP concentration; effect of Ca^{2+}. (**A**) Scatter plot showing the lifetimes of Myo1c at 5, 20 and 100 μM ATP (*n*=127, 427 and 97, respectively). Data are plotted as a cumulative distribution (e.g., data values that are less than each time point are scored and plotted at that time point) and normalised to 100%. Myo1c bound lifetime distribution exhibits a double exponential decay at all three ATP concentrations, suggesting that there are two exit routes from the attached state. Rate constants obtained by fitting a dual exponential decay indicate that there is a fast, ATP-independent phase and a slow, ATP-dependent phase (). (**B**) Lifetime distribution for data obtained at pCa 4.11 with 5 and 20 μM ATP plotted as before. The data for 20 μM ATP pCa 8 are replotted from (A) for comparison. The slow phase is accelerated by about 10-fold. (**C**) The relationship between step size estimate and duration of attachment is plotted by first ranking all event amplitudes on the basis of their attached lifetime and then calculating the running average of the lifetime and step size over a window of 80 consecutive data points (each estimate of mean step size for a given time window has a s.e.m. given by s.d./√*n*=14/8.9=1.5 nm). The data are fitted to an equation which assumes that the fast population produces no work stroke, whereas the slow population gives a work stroke (*d*_{max}) of 4.2 nm in the absence of calcium and 7.2 nm in the presence of calcium. The fitted line: Work stroke_{t}= *d*_{max} × *{ Ae*^{- rst} /(Ae^{- rst}+ *(1-A)e*^{- rft})}, where *A*_{slow}=relative amplitude of slow phase (%/100), *d*_{max} =work stroke produced by slow population, *r*_{s}=rate constant of slow phase, and *r*_{f}=rate constant of fast phase. See for fitting parameters. Note the similarity of fit parameters used for lifetime data plots (A) and amplitude data plots (C).

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