Fig. 2. Enzymatic and mechanical properties of wild-type and R403Q human β-cardiac sS1.
(A) Actin-activated ATPase activities (circles) of human wild-type (black) and R403Q mutant (red) β-cardiac sS1 at 23°C. The data points shown are the average of multiple experiments (n = 4 exp, 3 preps). The data are fit (solid lines, black for wild-type and red for R403Q) to the Michaelis-Menten equation to obtain the kcat and Km values. Errors reported are SEM. (B) Histograms of all single-molecule force events for wild-type (gray bars) (~2500 events, n = 23 exp, 10 preps) and R403Q human β-cardiac sS1 (red bars) (~1800 events, n = 10 exp, 4 preps). We used a Gaussian distribution function with a cutoff to take into account the missing small force events. The force distributions showed a long tail in the higher force regimes. These high-force events are a small fraction, but they can easily alter the fitting results and hence the interpretation of the data. To extract the mean force, we instead used a double Gaussian distribution (black line for wild type and red line for R403Q) and the fit results using the first Gaussian peak were used to bootstrap the data (see Optical trap setup and single-molecule measurements under Materials and Methods). For R403Q, the force distribution is shifted toward lower force values as compared to wild-type human β-cardiac sS1. (C) Distribution of mean forces obtained from bootstrapping of 300 single events from the total number of force events [as shown in (B), ~2500 events from n = 23 exp, 10 preps for wild type and ~1800 events from n = 10 exp, 4 preps for R403Q]. A Gaussian function was fit to the bootstrapped mean histogram (solid line, black for wild type and red for R403Q) to obtain a mean force value. The mean force of wild type and R403Q is significantly different from each other within 95% confidence interval. (D) Cumulative frequency distribution (solid line, black for wild type and red for R403Q) calculated from all single-molecule force events (~2500 events from n = 23 exp, 10 preps for wild type and ~1800 events from n = 10 exp, 4 preps for R403Q). Vertical dotted lines (black for wild type and red for R403Q) show a half-probability (shown as a black horizontal dashed line) of the force distribution, which is smaller for R403Q as compared to wild type.