Figure 4.
Single-molecule optical trapping: lifetime of AM interactions. A, the experimental setup for three-bead optical trapping measurements. Note that the indicated components, bead size, and protein dimensions are not to scale. B, original displacement over time data records; actin-myosin interaction can be observed as a reduction in the large thermal fluctuations from a bead at 10 μm ATP. The top and bottom panels show records collected from fast and slow WT myosin S1, respectively. Some of the interaction events are shown as green (fast WT) and red (slow WT) lines to indicate the duration of lifetimes, ton. Measurements were done by applying positive feedback and triangular wave (∼600 Hz). C–J, for WT-S1f, WT-S1s, and chimeras, event lifetimes (ton) are plotted in histograms and fitted with a single-exponential decay function to determine the average time constant. K, the bar diagram shows the average ton for WT and chimeric motors at 10 μm ATP concentration and room temperature of ∼22 °C. Error bars, S.E. (from the fits). For WT-S1f, n = 5000 events, N = 21; for WT-S1s; n = 1675 events, N = 19, for S1f-cgmRLC; n = 1517 events, N = 10, for S1f-MLC2B; n = 1953 events, N = 16, for S1f-MLC2v; n = 5714 events, N = 22, for S1s-cgmRLC; n = 1468 events, N = 12, for S1s-MLC2v; n = 400 events, N = 4, for S1s-MLC2B; n = 900 events, N = 8 (where N = number of individual motor molecules and n = number of AM interaction events). Statistical significance was calculated for the following pairs of motors using nonparametric Mann–Whitney U test: WT-S1f and WT-S1s, p < 0.00001; WT-S1s and S1s-cgmRLC, p < 0.0001; WT-S1s and S1s-MLC2B, p < 0.0001; WT-S1f and S1f-MLC2B, p = 0.0691; WT-S1f and S1f-MLC2v, p < 0.0001, WT-S1f and S1f-cgmRLC, p = 0.114; WT-S1s and S1s-MLC2v, p = 0.0941. p < 0.05 was considered statistically significant; p > 0.05 was considered not statistically significantly different.