Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C

The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium‐loaded (1H, 15N, and 13C) regulatory N‐domain of chicken skeletal troponin‐C (1–90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical‐shift information, along with detailed NOE analysis and 3JHNHα coupling constants, permitted the determination and quantification of the Ca2+‐induced secondary structural change in the N‐domain of TnC. For both structures, 5 helices and 2 short β‐strands were found, as was observed in the apo N‐domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761–779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix‐B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca2+‐induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in α‐helical content associated with the transition from apo to the 2Ca2+ state of the N‐domain of TnC. Therefore, the Ca2+‐induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.