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Results: 4

1.
Figure 3

Figure 3. From: Structural and Kinetic Effects of PAK3 phosphorylation mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament.

FRET-based Ca2+ titration of the changes of FRET between cTnI(160C)AEDANS and cTnC(89C)DDPM within the thin filament at different conditions. Black: from thin filament containing non-phosphorylated cTnI without strongly bound S1, red: from thin filament containing the pseudo-phosphorylation of cTnI(S151E) without the presence of strongly bound S1; blue: from thin filament containing non-phosphorylated cTnI in the presence of strongly bound S1, pink: from thin filament containing the pseudo-phosphorylation of cTnI(S151E) in the presence of strongly bound S1. The curves were fitted with the Hill equation to obtain values of pCa50 and the Hill coefficient. These parameters are listed in Table 2.

Yexin Ouyang, et al. J Mol Biol. ;400(5):1036-1045.
2.
Figure 4

Figure 4. From: Structural and Kinetic Effects of PAK3 phosphorylation mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament.

FRET-based kinetic tracings of normalized FRET changes between cTnI(160C)AEDANS and cTnC(89C)DDPM triggered by Ca2+ dissociation from cTnC in the reconstituted thin filaments containing non-phosphorylated cTnI (black dots) and the pseudo-cTnI(S151E) (black triangles). These two sets of kinetic tracings were obtained by averaging 7 or 8 stopped-flow tracings. The kinetic tracings were fitted with a two-exponential function (solid smooth lines), and recovered rate constants and amplitudes are given in Table 2.

Yexin Ouyang, et al. J Mol Biol. ;400(5):1036-1045.
3.
Figure 2

Figure 2. From: Structural and Kinetic Effects of PAK3 phosphorylation mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament.

Distance analysis of the time-resolved FRET data shown in panel B of Figure 1. A: Area normalized distribution of distance between residue Cys160 of cTnI and residue Cys89 of cTnC within the thin filament without the presence of the pseudo-phosphorylation of cTnI(S151E). B: Area normalized distribution of distance between residue Cys160 of cTnI and residue Cys89 of cTnC within the thin filament containing the pseudo-phosphorylation of cTnI(S151E). A value of κ2 = 2/3 was used in these calculations. Circle – thin filament samples, triangle – thin filament in the presence of strongly bound crossbridges, solid circle and triangle – samples in the absence of Ca2+ and the open circle and triangle – samples in the presence of Ca2+. All distributions are plotted using equation 3.

Yexin Ouyang, et al. J Mol Biol. ;400(5):1036-1045.
4.
Figure 1

Figure 1. From: Structural and Kinetic Effects of PAK3 phosphorylation mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament.

Donor (AEDANS) fluorescence spectra and fluorescence decays of FRET in the cardiac thin filament reconstituted with cTnI(160C)AEDANS and cTnC(89C)DDPM. A: Steady-state fluorescence spectra measured at different conditions with the reconstituted thin filament preparations without the presence of strongly bound crossbridges; B: Steady-state spectral measurements of the samples used in the panel A, but with the presence of strongly bound crossbridges; C: Fluorescence intensity decays of the samples used in the Panel A; and D: Fluorescence intensity decays of the samples used in the Panel B. Color codes: black – donor-only sample without Ca2+; dark red – donor-only sample plus Ca2+, red – donor-acceptor sample without Ca2+, pink - donor-acceptor sample plus Ca2+, light blue - donor-acceptor sample containing mimic phosphorylated cTnI without Ca2+, blue - donor-acceptor sample containing mimic phosphorylated cTnI plus Ca2+, and dark blue in Panel C and Panel D – profile of the excitation. Excitation wavelength was 343 nm, and the samples were in 50 mM Mops at pH 7.0, 1mM DTT, 1 mM EGTA, 5 mM MgCl2, 0.15 M KCl. When Ca2+ was present, it was at 2 mM CaCl2.

Yexin Ouyang, et al. J Mol Biol. ;400(5):1036-1045.

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