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 (H-1 and N-15) and calcium-loaded (H-1, N-15, and C-13) 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 (3)J(HNH alpha) 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 beta-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 2Ca(2+) 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 ct-helical content associated with the transition from apo to the 2Ca(2+) 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.