Fig. 5.

Functional properties of wild-type (WT) and mutant full-length PfMyoA constructs. a Schematic of constructs used for functional analysis. Ser19 is fully phosphorylated during protein expression in Sf9 cells (Supplementary Fig. 6). b Speed distributions from a representative in vitro motility assay. WT, 3.88 ± 0.54 µm/s; S19A, 2.07 ± 0.28 µm/s; K764E, 1.75 ± 0.22 µm/s; and ∆N, 0.23 ± 0.04 µm/s. Values, mean ± SD (Supplementary Table 3 shows data from multiple protein preparations). c Actin-activated ATPase activity for WT, V_max = 138 ± 4 s⁻¹ and K_m = 30.3 ± 2.3 µM; S19A, V_max = 74.0 ± 2.0 s⁻¹ and K_m = 8.5 ± 1.0 µM; K764E, V_max = 72.9 ± 1.9 s⁻¹ and K_m = 18.2 ± 1.4 µM; and ∆N, V_max = 9.13 ± 0.20 s⁻¹ and K_m = 7.34 ± 0.67 µM. Data from 2 protein preparations and 3 experiments for each construct were fitted to the Michaelis-Menten equation. Error, SE of the fit. d ADP release rates from acto-PfMyoA. WT, 334 ± 36 s⁻¹; S19A, 115.8 ± 10.9 s⁻¹; K764E, 103.5 ± 8.6 s⁻¹; ∆N, 10.32 ± 0.91 s⁻¹. Values, mean ± SD. WT vs. any other construct. p < 0.0001; S19A vs. K764E, NS; S19A or K764E vs. ∆N, p < 0.01 (one way ANOVA followed by a Tukey’s Honest Significant Difference post-hoc test). Data from at least 3 protein preparations of each construct at different temperatures are shown in Supplementary Table 4. e Ensemble force measurements using a utrophin-based loaded in vitro motility assay. A myosin that produces more force requires higher utrophin concentrations to arrest motion: WT, 1.40 ± 0.08 nM; S19A, 2.42 ± 0.17 nM; K764E, 3.04 ± 0.30 nM; ∆N, 10.8 ± 0.8 nM. Error, SE of the fit. Data from two protein preparations and three experiments for each construct. Supplementary Fig. 7b shows these force data and fits extended to higher utrophin concentrations. Supplementary Fig. 7c–e shows ∆N data shown with an expanded y-axis. Skeletal actin was used for all experiments. Temperature, 30 °C. Source data are provided as a Source Data file