Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnl). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca2+ activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca2+-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnl(L129W/SI51C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca2+-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca2+ titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin SI to AEDANS attached to Cys151 of cTnI was very small and Ca2+ independent. The present results show that the rate of Ca2+-induced structural transition and Ca2+ sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca2+ sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound SI significantly increased the Ca2+ sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca2+ sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by-adrenergic stimulation. (c) 2006 Elsevier Inc. All rights reserved.