Figure S6.
Effects of Tm-binding mutants on phosphorylation-dependent binding to actin–Tm and actin. Effects of Tm-binding mutants C0–C2 on actin–Tm and actin TR-F were tested with and without PKA treatment. Tm-binding mutants reverse charges (EASE; R215E/K218E) or introduce additional positive charges (RRKK; A216R/S217K) in the Tm-binding loop 215–218, RASK, of C0–C2. For comparison, curves for WT C0–C2 are included in each graph. (A) WT (black lines) and R215E/K218E (EASE in red) effects on IAEDANS–actin–Tm for unphosphorylated (solid lines) and phosphorylated (+PKA, dotted lines) C0–C2 from 0 to 20 µM cMyBP-C added. (B) Same conditions as A, except that IAEDANS–actin was used. (C and D) Zooming in on the lower concentrations (0 to 5 µM) cMyBP-C added in A and B. (E–H) The same conditions as A–D above but comparing WT (black lines) and A216R/S217K (RRKK in green). For the unphosphorylated Tm-binding mutant (charge reversal-EASE), apparent Kd changes trended toward significant (P = 0.11) for actin–Tm, but not for actin alone. The phosphorylated mutant did not fit well to a quadratic binding equation for either actin or actin–Tm (but did fit to a linear equation). For the unphosphorylated positive Tm-binding mutant (additional positive charges, RRKK) apparent Kd changes were significant (P < 0.05) for binding to both actin and actin–Tm. For phosphorylated RRKK binding, changes in Kd did not reach significance. Refer to Table S1 for statistical analysis of fitted binding properties for curves. See Table S2 for comparisons of binding at specific substoichiometric C0–C2 concentrations discussed in supplemental Results. Arrows in indicate the C0–C2 concentrations to be used in future screens (thick arrows, 1.25 µM for actin–Tm in A, C, E, and G; thin arrows, 2.5 µM for actin alone in B, D, F, and H). Data are provided as mean ± SE (n > 4).

Effects of Tm-binding mutants on phosphorylation-dependent binding to actin–Tm and actin. Effects of Tm-binding mutants C0–C2 on actin–Tm and actin TR-F were tested with and without PKA treatment. Tm-binding mutants reverse charges (EASE; R215E/K218E) or introduce additional positive charges (RRKK; A216R/S217K) in the Tm-binding loop 215–218, RASK, of C0–C2. For comparison, curves for WT C0–C2 are included in each graph. (A) WT (black lines) and R215E/K218E (EASE in red) effects on IAEDANS–actin–Tm for unphosphorylated (solid lines) and phosphorylated (+PKA, dotted lines) C0–C2 from 0 to 20 µM cMyBP-C added. (B) Same conditions as A, except that IAEDANS–actin was used. (C and D) Zooming in on the lower concentrations (0 to 5 µM) cMyBP-C added in A and B. (E–H) The same conditions as A–D above but comparing WT (black lines) and A216R/S217K (RRKK in green). For the unphosphorylated Tm-binding mutant (charge reversal-EASE), apparent Kd changes trended toward significant (P = 0.11) for actin–Tm, but not for actin alone. The phosphorylated mutant did not fit well to a quadratic binding equation for either actin or actin–Tm (but did fit to a linear equation). For the unphosphorylated positive Tm-binding mutant (additional positive charges, RRKK) apparent Kd changes were significant (P < 0.05) for binding to both actin and actin–Tm. For phosphorylated RRKK binding, changes in Kd did not reach significance. Refer to Table S1 for statistical analysis of fitted binding properties for curves. See Table S2 for comparisons of binding at specific substoichiometric C0–C2 concentrations discussed in supplemental Results. Arrows in indicate the C0–C2 concentrations to be used in future screens (thick arrows, 1.25 µM for actin–Tm in A, C, E, and G; thin arrows, 2.5 µM for actin alone in B, D, F, and H). Data are provided as mean ± SE (n > 4).

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