Krudy G A, Kleerekoper Q, Guo X, Howarth J W, Solaro R J, Rosevear P R
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
J Biol Chem. 1994 Sep 23;269(38):23731-5.
NMR spectroscopy and selective isotope labeling of both recombinant cardiac troponin C (cTnC3) and a truncated cardiac troponin I (cTnI/NH2) lacking the N-terminal 32-amino acid cardiac-specific sequence have been used to probe protein-protein interactions central to muscle contraction. Using [methyl-13C]Met-labeled cTnC3, all 10 cTnC Met residues of Ca(2+)-saturated cTnC3 could be resolved in the two-dimensional heteronuclear single- and multiple-quantum coherence spectrum of the cTnI.cTnC complex. Based on the known Met assignments in cTnC3, the largest chemical shift changes were observed for Met81, Met120, Met137, and Met157. Methionines 120, 137, and 157 are all located in the C-terminal domain of cTnC. Methionine 81 is located at the N terminus of the central helix. Minimal chemical shift changes were observed for Met45, Met47, and Met103 of cTnC3 in the cTnI.cTnC complex. All 6 Met residues in [methyl13C]Met-labeled cTnI/NH2 could be resolved in the cTnI.cTnC complex, suggesting that both cTnI and cTnC form a stable homogeneous binary complex under the conditions of the NMR experiment. In the absence of added protease inhibitors in the cTnI.cTnC complex, cTnI/NH2 was found to undergo selective proteolysis to yield a 5.5-kDa N-terminal fragment corresponding to residues 33-80. Judging from the NMR spectra of [methyl13C]Met-labeled cTnC3, cTnI-(33-80) was sufficient for interaction with the C-terminal domain of cTnC in a manner identical to that observed for native cTnI/NH2. However, in the presence of the proteolytic fragment cTnI-(33-80), the chemical shift of Met81 was not perturbed from its position in free cTnC3. Thus, residues located C-terminal to Arg80 in cTnI appear to be responsible for interaction with the N-terminal half of cTnC. Taken together, these results provide strong evidence for an antiparallel arrangement for the two proteins in the troponin complex such that the N-terminal portion of cTnI interacts with the C-terminal domain of cTnC. This interaction likely plays a role in maintaining the stability of the TnI.TnC complex.
核磁共振光谱法以及对重组心肌肌钙蛋白C（cTnC3）和缺少N端32个氨基酸心脏特异性序列的截短型心肌肌钙蛋白I（cTnI/NH2）进行选择性同位素标记，已被用于探究肌肉收缩核心的蛋白质-蛋白质相互作用。使用[甲基-13C]甲硫氨酸标记的cTnC3，在cTnI.cTnC复合物的二维异核单量子和多量子相干光谱中，可以分辨出Ca(2+)饱和的cTnC3的所有10个cTnC甲硫氨酸残基。基于cTnC3中已知的甲硫氨酸归属，观察到甲硫氨酸81、甲硫氨酸120、甲硫氨酸137和甲硫氨酸157的化学位移变化最大。甲硫氨酸120、137和157均位于cTnC的C端结构域。甲硫氨酸81位于中央螺旋的N端。在cTnI.cTnC复合物中，观察到cTnC3的甲硫氨酸45、甲硫氨酸47和甲硫氨酸103的化学位移变化最小。在cTnI.cTnC复合物中，可以分辨出[甲基13C]甲硫氨酸标记的cTnI/NH2中的所有6个甲硫氨酸残基，这表明在核磁共振实验条件下，cTnI和cTnC形成了稳定的均匀二元复合物。在cTnI.cTnC复合物中不添加蛋白酶抑制剂的情况下，发现cTnI/NH2会发生选择性蛋白水解，产生一个5.5 kDa的N端片段，对应于第33 - 80位残基。从[甲基13C]甲硫氨酸标记的cTnC3的核磁共振光谱判断，cTnI-(33 - 80)足以与cTnC的C端结构域以与天然cTnI/NH2相同的方式相互作用。然而，在存在蛋白水解片段cTnI-(33 - 80)的情况下，甲硫氨酸81的化学位移并未从其在游离cTnC3中的位置发生扰动。因此，cTnI中位于精氨酸80 C端的残基似乎负责与cTnC的N端一半相互作用。综上所述，这些结果为肌钙蛋白复合物中两种蛋白质的反平行排列提供了有力证据，即cTnI的N端部分与cTnC的C端结构域相互作用。这种相互作用可能在维持TnI.TnC复合物的稳定性中起作用。
