Calcium-dependent changes in the flexibility of the regulatory domain of troponin C in the troponin complex.
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.
Blumenschein
Tharin M A
TM
Canadian Institutes of Health Research Group in Structure and Function and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Stone
Deborah B
DB
Fletterick
Robert J
RJ
Mendelson
Robert A
RA
Sykes
Brian D
BD
D000818:Animals; D016880:Anisotropy; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D018360:Crystallography, X-Ray; D004533:Egtazic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D015233:Models, Statistical; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014336:Troponin / Q000737:chemistry; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism
D014336:Troponin; D019209:Troponin C; D004533:Egtazic Acid; D008274:Magnesium; D002118:Calcium