Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C

Figures

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  Fig 1. FRET-TnC constructs varying the FRET pair of fluorophores and linker length. Five constructs were generated by flanking human cardiac troponin C (cTnC) with one of three FRET pairs of fluorophores. Constructs also differ by the linker length (indicated by number of amino acids, aa) separating the fluorescent proteins from cTnC.

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  Fig 2. Schematic and crystal structure of cTnC. cTnC is shown in a schematic representation (gray, left) and the crystal structure (PDB 1J1E, right). Mutated residues (used in a subset of studies) are: D65 highlighted in orange at the N-terminus; and D104 and D140 highlighted in red and yellow, respectively, at the C-terminus.

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  Fig 3. Examples of individual fluorescence spectra obtained during end point titrations. Examples of spectra (A) for construct CTV-TnC (1μM) at 400μM Ca total, excited at various excitation wavelengths (λex; from purple to orange, in order: 400nm, 404nm, 408nm, 410nm, 414nm, 418nm, 420nm, 424nm, 433nm). (B) FRET ratios (FR, Eq 2) plotted as a function of λex for construct CTV-TnC (1μM), under the 3 conditions tested: initial condition 100μM EDTA (filled circles); next 400μM Ca (as in panel A) (open circles); and lastly 3mM EDTA (filled, inverted triangles). Note that FR is independent of λex and thus absolute value of acceptor emission intensity, and also that FR varies reversibly with divalent cation binding.

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  Fig 4. End point Ca2+-titrations comparing all FRET constructs in the absence or presence of 3mM Mg2+. In (A) the initial condition (100μM EDTA) represents apo-TnC, depleted of divalent cations, whereas in (C) the initial condition is TnC bound to ~2 Mg2+ achieved by adding 3mM Mg2+ to the starting buffer containing 100μM EGTA. Differences in FR (ΔFR) between the Ca2+ saturated condition and the average initial and final conditions were calculated (B) from the data in (A) in the absence of Mg2+, or (D) from the data in (C) in the presence of 3mM Mg2+. Asterisks indicate (A and C) that bracketed values are significantly different (t-test, p<0.05), or (B and D) that the value is significantly different from 0 (t-test, p<0.01). Plotted values represent mean + SE (N = 3).

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  Fig 5. Analytical ultracentrifugation (AUC) indicates global conformational changes of CTV-TnC upon divalent cation binding. The least compact structure is apo CTV-TnC (solid line). ~2Mg2+-CTV-TnC (dotted line) shows an intermediate conformation, whereas Ca2+ saturated CTV-TnC (dashed line) adopts the most compact conformation. Sedimentation coefficients are given in the text.

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  Fig 6. Ca2+-activated isometric force of skinned porcine cardiac muscle preparations reconstituted with WT-TnC or CTV-TnC. (A) Endogenous TnC was extracted (solid bars) and fibers were reconstituted with either WT-TnC or CTV-TnC (gray bars). The extent of extraction and reconstitution was defined by the percent of Ca2+-activated, steady state isometric force (pCa4) normalized to that measured prior to extraction of endogenous TnC. (B) Ca2+-sensitivity of steady state isometric force development of the two sets of reconstituted fibers was measured by incubation with different pCa solutions. Force was normalized to the maximum (pCa4) measured in the same preparation after reconstitution. Data were fit to the Hill relation (Eq 4). Plotted values represent mean + SE (N = 6).

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  Fig 7. Ca2+ and Mg2+ end point titrations of EF-hand-inactivation mutants of CTV-TnC. Mutants of CTV-TnC were generated by inactivating one or more EF hands (see Materials and Methods) to separate the contribution of the different EF hands to the total FRET signal. End point titrations with Ca2+ in the absence of Mg2+ (A, B) were performed with mutants as described for CTV-TnC WT in (Fig 4A and 4B). For comparison, FR and ΔFR for CTV-TnC WT were replotted (in A and B, respectively) from (Fig 4A and 4B). Panel (C) shows the effects of Mg2+ on FR. (D) shows ΔFR for data in C due to Mg2+ binding. Asterisks indicate (A and C) that bracketed values are significantly different (t-test, p<0.05), or (B and D) that the value is significantly different from 0 (t-test, p<0.01). Plotted values represent mean + SE (N = 3).

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  Fig 8. Ca2+ and Mg2+ titrations of CTV-TnC and CTV-Tn complex allow determination of EF hand affinities for divalent cations from FR. (A) Ca2+ titrations of CTV-TnC (1μM) were performed in the presence (solid symbols) or absence (open symbols) of 2mM Mg2+free to determine the affinities of sites III and IV (high affinity), and site II (low affinity) for Ca2+. (B) Mg2+ titration of CTV-TnC (1 μM), where the binding of Mg2+ is shown for sites III and IV. (C) Ca2+ titration of CTV-TnC reconstituted in Tn complex, in the presence of 2mM Mg2+free. Insets in (A) and (C) show expansion of the initial portion of the titrations for clarity. Data were fitted using nonlinear least squares regression to either a double-Hill relation (Eq 5) in (A and C), or a single Hill relation (Eq 6) in (B). All regression parameter estimates are given in Table 3. Plotted values represent mean + SE (N = 4).