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Arch Biochem Biophys. 2014 May 15;550-551:1-11. doi: 10.1016/j.abb.2014.03.013. Epub 2014 Apr 5.

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation.

Author information

  • 1The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA.
  • 2The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USA.
  • 3The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA; The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USA. Electronic address: wdong@vetmed.wsu.edu.

Abstract

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.

Copyright © 2014 Elsevier Inc. All rights reserved.

KEYWORDS:

Cardiac troponin T; FRET; PKC phosphorylation; Thin filament regulation

PMID:
24708997
[PubMed - indexed for MEDLINE]
PMCID:
PMC4059398
Free PMC Article
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