Calcium‐dependent protein–protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I

The goal of this study was to relate conformational changes in the N‐terminal domain of chicken troponin I (TnI) to Ca 2+ activation of the actin–myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl‐reactive pyrene‐containing fluorophores [ N ‐(1‐pyrene)maleimide, and N ‐(1‐pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca 2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca 2+ ‐dependent myofibrillar ATPase. The binding of Ca 2+ to the two N‐terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca 2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N‐terminal region of TnI may be necessary for full activation of muscle contraction.