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

Abstract

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 ³J_HNHα 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.