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 ('H and "N) and calcium-loaded ('H, IsN, 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 3JHNHa 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 0-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg 0, James MNG, 1988, JMol 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 a-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.Keywords: calcium; CD; NMR; regulatory domain of troponin-C; secondary structural change Troponin-C has a key role in muscle contraction of vertebrate striated muscle (skeletal and cardiac). The binding of Ca2+ to TnC induces a conformational change that affects the interaction between TnC, troponin-I (TnI), and troponin-T (TnT). This interaction blocks the inhibitory action of TnI, allowing formation of the Mg2+-activated ATPase actomyosin complex, and ultimately leads to muscle contraction. The roles and interactions of proteins in the regulatory system of striated muscle have been studied extensively (Leavis & Gergely, 1984; Ohtsuki et al.,

4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy

“…4); and (2) a N ( i , i ) / a N (i -1, i ) ratios that are significantly greater than 1 for residues in the a helix, and less than 1 for residues in &sheet regions. This is significant because this ratio tends to approach unity as the amount of spin diffusion increases (Gagne et al, 1994).…”

“…In these experiments, statistically, only NOEs between "N-attached protons within asinglesubunif are observed. These NOE data, summarized in Figure 5 , are categorized as strong, medium, or weak after normalization of the cross peak heights with respect to those of the corresponding NH diagonal resonance to correct for NH peak height differences (Gagne et al, 1994). Evidence against significant spin-diffusion effects in the 50-ms NOESY spectra includes: (1) the absence of NN (i, i + 3) correlations in the a-helical region (Fig.…”

The solution structure of human endothelin‐2 a 1H‐NMR and CD study

“…The N-terminal Ser2ϪSer5 had coupling constants of 8.0 Hz or more, which was also indicated by high [47]. Grey bars indicate local secondary structure based on a ratio of NOE intensities [44]. Positive and negative bars denote β-conformation and helix, respectively.…”

“…These restraints were derived from the ratio of the d AN (i,iϩ1) and d NA (iϩ1,iϩ1) NOE volumes [44]. Ratios could be calculated for 20 residues, but were only used if (a) the volumes were not suspected to be unequally affected by solvent signal suppression, (b) the NOEs showed no overlap and (c) the corresponding distance ratio differed from 1 by more than 2.5 %, which resulted in 16 dihedral ψ-angle intervals.…”

“…The allowed generous intervals were Ϫ60Ϯ 140°and 120 Ϯ100°for volume ratios Ͼ 1 and Ͻ 1, respectively, as determined by a strict interpretation of Fig. 3 in [44].…”