Effects of WT and R282W HCM mutant on phosphorylation-modulated binding to actin at submaximal phosphorylation by PKA. (A) TR-F of IAEDANS–actin incubated with increasing concentrations of C0–C2 (WT, black; R282W, blue) either unphosphorylated (solid lines) or phosphorylated using 7.5 ng PKA/µg C0–C2 (dashed lines). For B–D, effects of HCM mutant R282W on C0–C2 phosphorylation were tested over a range of PKA levels (0–5 ng PKA/µg C0–C2). (B) SYPRO Ruby (total protein; top bands) and Pro-Q Diamond (phosphorylated protein; bottom bands) stains of SDS-PAGE. (C) Relative phosphorylation levels of WT and R282W (normalized to the ratio of the Pro-Q Diamond/SYPRO Ruby intensities for WT C0–C2 at 5 ng PKA/µg C0–C2). Phosphorylation levels of R2828W are significantly different (P < 0.00006) from WT for all concentrations of PKA. (D) WT and R282W effects on IAEDANS–actin lifetime change as a function of PKA concentration. At intermediate phosphorylation levels (0.5 and 1.5 ng PKA/µM C0–C2), binding to actin detected by TR-F is significantly different between WT and R282W (*, P < 0.003). Average data are provided as mean ± SE (n > 4).