Abstract
With the recent advances in structure determination of the troponin complex, it becomes even more important to understand the dynamics of its components and how they are affected by the presence or absence of Ca(2+). We used NMR techniques to study the backbone dynamics of skeletal troponin C (TnC) in the complex. Transverse relaxation-optimized spectroscopy pulse sequences and deuteration of TnC were essential to assign most of the TnC residues in the complex. Backbone amide (15)N relaxation times were measured in the presence of Ca(2+) or EGTA/Mg(2+). T(1) relaxation times could not be interpreted precisely, because for a molecule of this size, the longitudinal backbone amide (15)N relaxation rate due to chemical shift anisotropy and dipole-dipole interactions becomes too small, and other relaxation mechanisms become relevant. T(2) relaxation times were of the expected magnitude for a complex of this size, and most of the variation of T(2) times in the presence of Ca(2+) could be explained by the anisotropy of the complex, suggesting a relatively rigid molecule. The only exception was EF-hand site III and helix F immediately after, which are more flexible than the rest of the molecule. In the presence of EGTA/Mg(2+), relaxation times for residues in the C-domain of TnC are very similar to values in the presence of Ca(2+), whereas the N-domain becomes more flexible. Taken together with the high flexibility of the linker between the two domains, we concluded that in the absence of Ca(2+), the N-domain of TnC moves independently from the rest of the complex.
Highlights
The troponin (Tn)1 complex is responsible for the regulation of contraction in striated skeletal and cardiac muscles
We compared the independent effects of TROSY and deuteration on the 55-kDa skeletal troponin complex by acquiring {1H,15N} HSQC and {1H,15N} TROSY-HSQC two-dimensional NMR spectra of troponin complex containing uniformly deuterated troponin I (TnI) and troponin T (TnT)-T2 and either 13C,15N- or 2H,13C,15N-labeled troponin C (TnC) in the presence of Ca2ϩ
The chemical shift comparisons imply that the N-domain of TnC has a similar structure when in the ternary troponin complex or in a binary complex with the peptide TnI-(115–131) (31), whereas the C-domain of TnC has a similar structure in the ternary troponin or in a binary complex with the peptide TnI(1– 40) (30)
Summary
The troponin (Tn) complex is responsible for the regulation of contraction in striated skeletal and cardiac muscles. Recent determination of the crystal structure of cardiac troponin (5) has revealed an apparently quite flexible complex. The sites in the N-domain are Ca2ϩspecific and important for the regulation of muscle contraction, whereas the EF-hand sites in the C-domain have higher affinity, can bind both Ca2ϩ and Mg2ϩ, and are permanently occupied with one of the two ions. Because of these differences, the two domains are known as regulatory and structural domains, respectively. Structures of complexes between TnC domains and TnI peptides (13–17) have revealed the nature of these interactions at atomic detail and have been confirmed by the recent x-ray structures of cardiac (5) and skeletal troponin (18)
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