Sheng Z L, Francois J M, Hitchcock-DeGregori S E, Potter J D
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
J Biol Chem. 1991 Mar 25;266(9):5711-5.
To investigate the role of the central helix of skeletal muscle troponin C (TnC), five deletion mutants (Dobrowolski, Z., Xu, G.Q., and Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710) of chicken TnC in the D/E linker region (K87EDAKGKSEEE97), dEDA, dKG, dKGK, dSEEE, and dKED-AKGK, were assayed for their ability to regulate muscle contraction by testing their effectiveness in restoring force and Ca2+ regulation to TnC-depleted rabbit skinned skeletal muscle fibers. By comparison with rabbit skeletal TnC, wild-type TnC, and chicken TnC, all mutants except dKG equally restored force development and Ca2+ regulation to TnC-depleted skinned muscle fibers. In contrast, approximately 4 times more dKG than rabbit skeletal TnC was required to reach 50% force restoration. Also, the pCa50 for dKG activation of force was significantly decreased. Thus, most of the TnC mutants that we studied did not have significantly altered biological activity in the skinned fiber assay. However, the 2-residue deletion in the central helix (dKG) significantly affected TnC activity. This deletion would be expected to produce a 160 degree rotation in the alpha-helix versus 60 degrees for dKGK and dEDA, 40 degrees in dSEEE, and 20 degrees in dKEDAKGK. Therefore, the change in orientation of the two Ca2(+)-binding domains appears to be a major parameter affecting TnC activity. The shift in the Ca2+ dependence in force activation may result from the inability of the Ca2(+)-specific domain to properly interact with its binding site on troponin I, an interaction which is known to increase the affinity of TnC for Ca2+ (Potter, J.D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633). In addition, the length of the central helix of TnC, Gly92, and the negatively charged cluster, EEE, appear not to be crucial for TnC activity.
为研究骨骼肌肌钙蛋白C（TnC）中心螺旋的作用，我们对鸡TnC在D/E连接区（K87EDAKGKSEEE97）的五个缺失突变体（Dobrowolski, Z., Xu, G.Q., and Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703 - 5710），即dEDA、dKG、dKGK、dSEEE和dKED - AKGK，通过检测它们恢复TnC缺失的兔去皮肤骨骼肌纤维力和Ca²⁺调节能力的有效性，来测定其调节肌肉收缩的能力。与兔骨骼肌TnC、野生型TnC和鸡TnC相比，除dKG外的所有突变体均能同等程度地恢复TnC缺失的去皮肤肌肉纤维的力产生和Ca²⁺调节能力。相反，达到50%力恢复所需的dKG量约为兔骨骼肌TnC的4倍。此外，dKG激活力的pCa50显著降低。因此，我们研究的大多数TnC突变体在去皮肤纤维实验中生物活性没有显著改变。然而，中心螺旋中的2个残基缺失（dKG）显著影响了TnC活性。预计该缺失会使α - 螺旋产生160度旋转，而dKGK和dEDA为60度，dSEEE为40度，dKEDAKGK为20度。因此，两个Ca²⁺结合结构域方向的改变似乎是影响TnC活性的主要参数。力激活中Ca²⁺依赖性的改变可能是由于Ca²⁺特异性结构域无法与其在肌钙蛋白I上的结合位点正确相互作用，已知这种相互作用会增加TnC对Ca²⁺的亲和力（Potter, J.D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628 - 4633）。此外，TnC中心螺旋的长度、Gly92以及带负电荷的簇EEE似乎对TnC活性并不关键。
