Figure 1.
Expression of recombinant human β-cardiac S1 and thin filament proteins.
(A) Lane 1, an Invitrogen Benchmark Ladder; Lane 2, WT human β-cardiac S1 (∼120 kDa) with a C-terminal GFP tag and the FLAG-tagged human ventricular essential light chain (ELC) (∼24 kDa). (B) Lane 1, an Invitrogen Benchmark Ladder; Lane 2, chicken skeletal actin (∼42 kDa); Lane 3, bovine tropomyosin (Tm; α isoform lower and β isoforms upper, ∼33 kDa); and Lane 4, human cardiac troponin complex (Tn; TnT ∼35 kDa, TnI ∼24 kDa, TnC ∼18 kDa). (C) A model of the troponin complex which is built from partial crystal structures of the human cardiac troponin complex of TnT (green), TnI (blue), and TnC (red) (Adapted from [72], PDB 1J1E). Mutations are shown in magenta. (D) Human cardiac TnT exons and functional domains [16]. HCM-causing mutations are in red and DCM-causing mutations in blue.
Figure 2.
Binding of troponin complex to pyrene-labeled tropomyosin at 23°C.
Each curve is the average of ≥ three individual curves, and error bars represent SEM. Buffer conditions were 20 mM Hepes, pH 7.5, 100 mM KCl, 2 mM MgCl2, 1 mM DTT. WT is in black, I79N in orange, R141W in green, E163K in red, and R173W in blue.
Table 1.
Summary of the maximal Ca2+ activated in vitro motility velocities and ADP release rates at 23°C with human β-cardiac S1.
Figure 3.
Ca2+ sensitivity of WT and mutant TnT thin filaments.
(A) Ca2+ sensitivity of thin-filament-activated ATPase activity of human β-cardiac S1 at 30°C. Each curve is the average of >10 individual curves, and error bars represent SEM. The curves were individually fit to the Hill equation, and normalized to the maximum activity of WT, as summarized in Table 2. WT is in black, I79N in orange, R141W in green, E163K in red, and R173W in blue. The shaded gray region represents the approximate physiological Ca2+ range during a heartbeat (See Discussion). (B) The effective Kd for Ca2+ induced conformation changes with (filled circles) and without (empty circles) cycling S1 using ANS-TnCT53C thin filaments at 23°C. Error bars represent SEM. The dotted line represents the average effective Kd for WT thin filaments. There was no significant difference with and without cycling motor (p>0.05). (C) The effective Ca2+ dissociation rates or koff with (filled circles) and without (empty circles) cycling S1 using ANS-labeled thin filament at 23°C. Error bars represent SEM. The dotted line represents the average effective koff for WT thin filaments. As with the Kd's, there was no significant difference in the effective koff with and without cycling motor (p>0.05).
Table 2.
Summary of the thin filament activated ATPase pCa curves for human β-cardiac S1 at 30°C.
Table 3.
Summary of Ca2+ sensitivity and properties of WT troponin complex at 23°C at various conditions.
Figure 4.
WT ANS-labeled troponin complex properties.
Ca2+ sensitivity of WT-ANS troponin complex and thin filament with different amounts of human β-cardiac S1 at 23°C. Each curve is the average of at least three individual curves, and error bars represent SEM. Black filled circles, thin filament with no S1; open circles, 0.1∶1 cycling S1 to thin filament; dark grey filled circles, 0.01∶1 rigor S1 to thin filament; light grey filled circles, 0.1∶1 rigor S1 to thin filament; open triangles, 1.5∶1 rigor S1 to thin filament; open squares, troponin (Tn) complex with no S1. TF in the legend stands for thin filament.