Abstract
Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using Förster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states.
Highlights
The contractile state of cardiac muscle is regulated by a series of Ca2ϩ and cross-bridge-dependent interactions among the thin filament proteins, including the subunits of troponin (Tn),1 tropomyosin, and actin
Cardiac muscle activation is initiated by the binding of Ca2؉ to the single N-domain regulatory site of cardiac muscle troponin C
We recently reported that in fully reconstituted cTn, Ca2ϩ binding to cardiac muscle troponin C (cTnC) causes a 9-Å increase in the length of cardiac muscle troponin I (cTnI)-I [7]
Summary
Sample Preparation—Wild type recombinant chicken slow skeletal TnC (identical in sequence to mouse cTnC) and an adult rat cTnT mutant, in which both endogenous tryptophan at positions 237 and 288 were converted into phenylalanine, were overexpressed in Escherichia coli BL21(DE3) (Invitrogen) and purified as previously described [7,8,9]. FRET measurement requires data collection from donor-only and donor-acceptor samples. The reduced chi squares ratio (R2 ) was used to judge goodness of fit for the distribution Anisotropy decays of both donor and acceptor were determined by measuring the emission polarized in the vertical and horizontal directions with vertically polarized excitation. For all time-resolved FRET measurements, the donor, tryptophan, was selectively excited at 295 nm, and its emission was collected near its 333 nm maximum with a 340-nm interference filter. The donor-acceptor overlap integral was determined for each probe pair under each experimental condition. The overlap integrals and the donor-only quantum yields enabled calculation of the Forster distance (Ro) for each experimental condition (Mg2ϩsaturating, Ca2ϩ-saturating, and denatured). Mg2ϩ- and Ca2ϩ-saturated models were optimized to near zero energy. The reported models are representative of three different energy-minimized structures
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