Dynamics of the C-terminal region of TnI in the troponin complex in solution
Tharin M. A. Blumenschein 1, Deborah B. Stone 2, Robert J. Fletterick 2, Robert A. Mendelson 2 and Brian D. Sykes 1*
1 University of Alberta
2 University of California, San Francisco
* To whom correspondence should be addressed. E-mail: brian.sykes{at}ualberta.ca.
Submitted on October 17, 2005
Revised on November 28, 2005
Accepted on 27 December 2005
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Abstract |
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The determination of crystal structures of the troponin complex (Takeda et al., Nature 424, 35; Vinogradova et al., Proc. Natl. Acad. Sci. USA 102, 5038) has advanced knowledge of the regulation of muscle contraction at the molecular level. However, there are domains important for actin binding that are not visualized. We present evidence that the C-terminal region of troponin I (TnI - residues 135-182) is flexible in solution and has no stable secondary structure. We use NMR spectroscopy to observe the backbone dynamics of skeletal [2H, 13C, 15N]-TnI in the troponin complex, in the presence of Ca2+ or EGTA/Mg2+. Residues in this region give stronger signals than the remainder of TnI, and chemical shift index values indicate little secondary structure, suggesting a very flexible region. This is confirmed by NMR relaxation measurements. Unlike TnC and other regions of TnI in the complex, the C-terminal region of TnI is not affected by Ca2+ binding. Relaxation measurements and reduced spectral density analysis are consistent with the C-terminal region of TnI being a tethered domain, connected to the rest of the troponin complex by a flexible linker, residues 137-146, followed by a collapsed region with at most nascent secondary structure.
Key Words:
NMR relaxation, chemical shift index, muscle contraction, muscle regulatory proteins, spectral density analysis, thin filament
