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Effects of mutations in the central helix of troponin C on its biological activity
Z L Sheng1, J M Francois, S E Hitchcock-DeGregori
1Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Abstract:
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.
Insights
Investigating skeletal muscle troponin C (TnC) central helix mutations, researchers found that most deletions did not alter muscle contraction. However, the dKG deletion significantly impaired TnC activity and Ca2+ regulation.
Area of Science:
- Muscle physiology and biophysics
- Protein structure-function relationships
- Calcium signaling in muscle contraction
Background:
- Skeletal muscle contraction is regulated by the troponin complex, particularly troponin C (TnC).
- The central helix of TnC plays a role in its interaction with other proteins and calcium binding.
- Understanding TnC structure-function is crucial for elucidating muscle regulatory mechanisms.
Purpose of the Study:
- To investigate the functional significance of the central helix in skeletal muscle troponin C (TnC).
- To assess the impact of specific TnC deletion mutants in the D/E linker region on muscle force and calcium regulation.
- To determine how alterations in the central helix orientation affect TnC's biological activity.
Main Methods:
- Creation of five deletion mutants of chicken TnC in the D/E linker region.
- Assay of mutant TnC effectiveness in restoring force and Ca2+ regulation to TnC-depleted rabbit skinned skeletal muscle fibers.
- Comparison of mutant TnC activity with wild-type and rabbit skeletal TnC.
Main Results:
- Most TnC deletion mutants (dEDA, dKGK, dSEEE, dKED-AKGK) equally restored force and Ca2+ regulation compared to wild-type.
- The dKG mutant, with a 2-residue deletion in the central helix, significantly impaired force development and Ca2+ regulation.
- The dKG mutant required four times more protein for 50% force restoration and showed a decreased pCa50 for force activation.
Conclusions:
- The orientation of TnC's two Ca2+-binding domains, influenced by central helix structure, is critical for TnC activity.
- The dKG deletion likely alters the interaction between TnC's Ca2+-specific domain and troponin I, affecting Ca2+ affinity.
- The length of the central helix and the negatively charged cluster (EEE) are not essential for TnC activity.