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.
Regulation of cardiac muscle function is initiated by binding of Ca2+ 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 Ca2+ 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 Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ 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 Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ 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.
Dong
Wen-Ji
WJ
The School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington 99164, USA. wdong@vetmed.wsu.edu
Jayasundar
Jayant James
JJ
An
Jianli
J
Xing
Jun
J
Cheung
Herbert C
HC
D008841:Actin Cytoskeleton; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004735:Energy Transfer; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D017550:Spectroscopy, Fourier Transform Infrared; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism*; D019210:Troponin I / Q000378:metabolism*
D000199:Actins; D019209:Troponin C; D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium