Journal article
FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation
Archives of biochemistry and biophysics, Vol.550-551, pp.1-11
05/15/2014
Handle:
https://hdl.handle.net/2376/109592
PMCID: PMC4059398
PMID: 24708997
Abstract
•cTnT phosphorylation by PKC was mimicked in in vitro thin filaments.•I–C switching was monitored by FRET for these phospho-mimics.•Phospho-mimics altered I–C switching Ca2+-sensitivity, structure, and kinetics.•cTnT(T204E) significantly reduced Ca2+-sensitivity of I–C switching.•cTnT(3M) and (4M) increased Ca2+-sensitivity, reduced I–C separation and kinetics.
FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca2+ and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca2+ 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 Ca2+ sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca2+-sensitivity. The rate of Ca2+-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 Ca2+-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca2+-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca2+-sensitivity and likely play an important role in cardiac regulation.
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Details
- Title
- FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation
- Creators
- William Schlecht - The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USAZhiqun Zhou - The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USAKing-Lun Li - The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USADaniel Rieck - The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USAYexin Ouyang - The Department of Integrated Neuroscience and Physiology, Washington State University, Pullman, WA 99164, USAWen-Ji Dong - The Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA 99164, USA
- Publication Details
- Archives of biochemistry and biophysics, Vol.550-551, pp.1-11
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Publisher
- Elsevier Inc
- Identifiers
- 99900547114801842
- Language
- English
- Resource Type
- Journal article