Journal article
Förster Resonance Energy Transfer Structural Kinetic Studies of Cardiac Thin Filament Deactivation
The Journal of biological chemistry, Vol.284(24), pp.16432-16441
06/12/2009
Handle:
https://hdl.handle.net/2376/106741
PMCID: PMC2713534
PMID: 19369252
Abstract
Cardiac thin filament deactivation is initiated by Ca
2+
dissociation from troponin C (cTnC), followed by multiple structural changes of thin filament proteins. These structural transitions are the molecular basis underlying the thin filament regulation of cardiac relaxation, but the detailed mechanism remains elusive. In this study Förster resonance energy transfer (FRET) was used to investigate the dynamics and kinetics of the Ca
2+
-induced conformational changes of the cardiac thin filaments, specifically the closing of the cTnC N-domain, the cTnC-cTnI (troponin I) interaction, and the cTnI-actin interaction. The cTnC N-domain conformational change was examined by monitoring FRET between a donor (AEDANS) attached to one cysteine residue and an acceptor (DDPM) attached the other cysteine of the mutant cTnC(L13C/N51C). The cTnC-cTnI interaction was investigated by monitoring the distance changes from residue 89 of cTnC to residues 151 and 167 of cTnI, respectively. The cTnI-actin interaction was investigated by monitoring the distance changes from residues 151 and 167 of cTnI to residue 374 of actin. FRET Ca
2+
titrations and stopped-flow kinetic measurements show that different thin filament structural transitions have different Ca
2+
sensitivities and Ca
2+
dissociation-induced kinetics. The observed structural transitions involving the regulatory region and the mobile domain of cTnI occurred at fast kinetic rates, whereas the kinetics of the structural transitions involving the cTnI inhibitory region was slow. Our results suggest that the thin filament deactivation upon Ca
2+
dissociation is a two-step process. One step involves rapid binding of the mobile domain of cTnI to actin, which is kinetically coupled with the conformational change of the N-domain of cTnC and the dissociation of the regulatory region of cTnI from cTnC. The other step involves switching the inhibitory region of cTnI from interacting with cTnC to interacting with actin. The latter processes may play a key role in regulating cross-bridge kinetics.
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Details
- Title
- Förster Resonance Energy Transfer Structural Kinetic Studies of Cardiac Thin Filament Deactivation
- Creators
- Jun Xing - theJayant J Jayasundar - From theYexin Ouyang - From theWen-Ji Dong - From the
- Publication Details
- The Journal of biological chemistry, Vol.284(24), pp.16432-16441
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Publisher
- American Society for Biochemistry and Molecular Biology; 9650 Rockville Pike, Bethesda, MD 20814, U.S.A
- Grant note
- HL80186 / National Institutes of Health
- Identifiers
- 99900546858101842
- Language
- English
- Resource Type
- Journal article