Troponin Complex Conformation Changes Upon Calcium Binding and Phosphorylation

Striated muscle contraction is regulated by troponin complex which consist of three subunits: troponin C (TnC) binds to calcium, troponin I (TnI) inhibits actomyosin ATPase activity and troponin T (TnT) anchors the complex to the thin filament. An increase in calcium concentration induces conformational changes in TnI and TnC. These changes are believed to result in the repositioning of tropomyosin (Tm), exposing myosin binding sites on actin and eventually leading to force production. The N-terminal extension (30-33 a.a) of the cardiac TnI subunit contains two PKA phosphorylation sites (serine 23/24). In the ON-state (+Ca2+), the position of the cTnI switch peptide (150-159) is in close proximity to the N-lobe of TnC whereas in the OFF-state (-Ca2+) its location is unknown. One of many possibilities is that the peptide is close to the coiled-coil region of cTnT (226-275) and cTnI (90-136). We designed mutants with labels on the N-lobe of TnC, the TnI/TnT coiled coil and at either end of the switch peptide to probe the ON/OFF states conformational changes of the switch peptide within the troponin complex. Conventional dipolar EPR and Double Electron-Electron Resonance (DEER) were used for distance measurements between the different mutants in the reconstituted troponin complex. For the ON-state the measured distance is in agreement with the crystal structure (Takeda et al. 2003). In the OFF-state we concluded that the switch peptide is found at two different positions a) close to the N-lobe of TnC and b) close to the TnI/TnT coiled-coil. Cardiac muscle regulation is modulated by the phosphorylation of serines (23/24) in the N-terminal extension. A model of the bisphosphorylated N-terminal extension suggests that phosphorylation induces the movement of the N-terminal extension toward the inhibitory region. We measured the relative distance in both unphosphorylated and bisphosphorylated states between N-terminal extension and the inhibitory region (TnI9/142) in the presence of calcium. Our results showed no distance change between un-&-bisphosphorylated states of TnI9/142.