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Updated: Jun 24, 2026
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Published on: October 2, 2021
Förster resonance energy transfer structural kinetic studies of cardiac thin filament deactivation
Jun Xing1, Jayant J Jayasundar2, Yexin Ouyang2
1Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294.
Abstract:
Cardiac thin filament deactivation is initiated by Ca2+ 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 Ca2+-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 Ca2+ titrations and stopped-flow kinetic measurements show that different thin filament structural transitions have different Ca2+ sensitivities and Ca2+ 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 Ca2+ 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.
Insights
Cardiac relaxation involves complex protein structural changes after calcium (Ca2+) leaves troponin C (cTnC). This study reveals a two-step deactivation process, detailing protein interactions crucial for heart muscle relaxation.
Area of Science:
- Cardiovascular Physiology
- Molecular Biology
- Biophysics
Background:
- Cardiac muscle relaxation is regulated by thin filament proteins, a process initiated by calcium dissociation from troponin C (cTnC).
- The precise molecular mechanisms and kinetics of these structural transitions during cardiac deactivation remain incompletely understood.
- Understanding these dynamics is crucial for elucidating the regulation of cardiac contractility and relaxation.
Purpose of the Study:
- To investigate the dynamics and kinetics of Ca2+-induced conformational changes in cardiac thin filaments using Förster resonance energy transfer (FRET).
- To elucidate the specific interactions between cTnC, troponin I (cTnI), and actin during cardiac deactivation.
- To determine the Ca2+ sensitivity and kinetics of distinct structural transitions within the cardiac thin filament.
Main Methods:
- Utilized FRET to monitor conformational changes in cTnC (N-domain closing) and interactions between cTnC-cTnI and cTnI-actin.
- Employed FRET Ca2+ titrations and stopped-flow kinetic measurements to analyze the dynamics.
- Used specifically engineered cysteine residues on cTnC and cTnI for FRET probe attachment.
Main Results:
- Different structural transitions within the cardiac thin filament exhibit distinct Ca2+ sensitivities and dissociation kinetics.
- Transitions involving the cTnI regulatory and mobile domains occur rapidly, while those involving the cTnI inhibitory region are slow.
- Ca2+ dissociation triggers a two-step deactivation process involving rapid cTnI-actin binding and slower switching of the cTnI inhibitory region.
Conclusions:
- Cardiac thin filament deactivation is a sequential process initiated by rapid structural rearrangements and followed by slower inhibitory region repositioning.
- The rapid binding of cTnI's mobile domain to actin, coupled with cTnC N-domain changes, drives initial deactivation.
- The slow transition of cTnI's inhibitory region to interact with actin is critical for regulating cross-bridge kinetics and cardiac relaxation.
