Journal article
Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments
Biochemistry (Easton), Vol.46(34), pp.9752-9761
08/28/2007
Handle:
https://hdl.handle.net/2376/113698
PMCID: PMC2547119
PMID: 17676764
Abstract
Regulation of cardiac muscle function is initiated by binding of Ca
2+
to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine tuned by phosphorylation of cTnI. The objective of the present study is to use a new Förster Resonance Energy Transfer-based structural marker to distinguish structural and kinetic effects of Ca
2+
binding, crossbridge interaction and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin and actin. Changes in the distance between Cys13 and Cys51 induced by Ca
2+
binding/dissociation were determined by FRET-sensed Ca
2+
titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca
2+
and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca
2+
sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca
2+
sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.
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Details
- Title
- Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments
- Creators
- Wen-Ji Dong - The School of Chemical Engineering and Bioengineering andJayant James Jayasundar - The Department of Veterinary and Comparative Anatomy Pharmacology and Physiology, Washington State University, Pullman, WA 99164Jianli An - The Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294Jun Xing - The Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294Herbert C Cheung - The Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294
- Publication Details
- Biochemistry (Easton), Vol.46(34), pp.9752-9761
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Identifiers
- 99900547656201842
- Language
- English
- Resource Type
- Journal article