Förster resonance energy transfer structural kinetic studies of cardiac thin filament deactivation.
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.
Xing
Jun
J
Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294.
Jayasundar
Jayant J
JJ
From the School of Chemical Engineering and Bioengineering, Pullman, Washington 99164.
Ouyang
Yexin
Y
From the School of Chemical Engineering and Bioengineering, Pullman, Washington 99164.
Dong
Wen-Ji
WJ
From the School of Chemical Engineering and Bioengineering, Pullman, Washington 99164; Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington 99164. Electronic address: wdong@vetmed.wsu.edu.
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D016296:Mutagenesis; D009206:Myocardium / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D019209:Troponin C; D019210:Troponin I; D002118:Calcium