Dynamics of a Troponin Chimera Reproduce the Effects of Calcium on the Troponin Complex

Abstract

In striated muscle, contraction is regulated in a Ca2+-dependent manner by the three subunits of the troponin complex. Troponin I (TnI) inhibits actomyosin ATPase in the absence of calcium; Ca2+ binding to troponin C (TnC) causes conformational changes that alter the interaction between TnI and TnC, removing the inhibition. These conformational changes are transmitted to the rest of the thin filament through interactions with troponin T (TnT). Nuclear magnetic resonance (NMR) studies of the core skeletal troponin complex (52 kDa) showed Ca2+-dependent changes in relaxation parameters of the regulatory region of TnC (residues 1-91) (Blumenschein et al., J. Biol. Chem. 280, 21924), and that the last 50 residues of TnI are disordered irrespective of the presence of calcium (Blumenschein et al., Biophys. J. 90, 2436). This disorder is postulated to be essential for muscle regulation (Hoffmann et al., J. Mol. Biol. 361, 625). Due to the size of the troponin complex, it was not possible to observe the remaining residues of the regulatory region of TnI (98-182). A chimeric polypeptide, containing the regulatory regions of TnI and TnC connected by a short linker (GGAGG), is capable of regulating actomyosin ATPase (Tiroli et al., FEBS Journal 272, 779), and at 20 kDa, provides a better target for NMR studies and the ability to visualise the residues so far unobserved. NMR relaxation measurements were used to study the dynamics of this troponin chimera in the presence and absence of calcium, and when the difference in molecular weight is taken into account, the relaxation parameters reproduce perfectly the results observed for the whole complex, in the presence and absence of calcium, both for the TnC and TnI regions previously observed.