Dynamics of a Skeletal Troponin C - Troponin I Chimera Probed by Comparison of Experimental and Simulated NMR Relaxation Parameters

Abstract

The activation of skeletal and cardiac muscle is triggered by the release of calcium from the sarcoplasmic reticulum. The calcium sensor is the troponin complex that is formed by three subunits: the calcium-binding protein troponin C (TnC), the inhibitory protein troponin I (TnI) and the tropomyosin-associated protein troponin T (TnT). When calcium binds to TnC, the resulting conformational change allows TnC to bind TnI, leading to the removal of the C-terminal region of TnI from actin. Consequential movement of the tropomyosin allows the binding of the myosin head to actin resulting in a power stroke. Regions of these proteins are highly flexible and the importance of these intrinsically disordered sections has been recently recognized and rationalized (Hoffman et al. J. Mol. Biol. 2006 361:625-633).Structural studies of the muscle system have been very successful in determining the structural organization of most of the molecular components involved in force generation at the atomic level. Although mainly α-helical, the structure and dynamics of TnI remains controversial, particularly in its C-terminal region. Different structures have been presented for this region: a single α-helix observed by x-ray crystallography, a “mobile domain” containing a small β-sheet derived from NMR restraints, and a mainly unstructured region according to NMR relaxation data. To investigate this, we have constructed a skeletal TnC-TnI chimera that contains the N-domain of TnC (1-91), a short linker (GGAGG), and the C-terminal region of TnI (98-182). Our objective is to determine which of the three proposed structures best fit the experimental 15N relaxation data for this chimera. The comparison between experimental and NMR relaxation parameters calculated from molecular dynamic simulations will be presented to assess the validity of the three models.