Förster Resonance Energy Transfer Structural Kinetic Studies of Cardiac Thin Filament Deactivation

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 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.Cardiac muscle utilizes troponin to sense the concentration changes of myoplasmic Ca 2ϩ and translate the transient Ca 2ϩsignal into a cascade of events within the thin filament that ultimately leads to force generation or relaxation. The cardiac thin filament is composed of the heterotrimeric troponin complex and tropomyosin bound to the double helical actin filament (1, 2). The cardiac troponin is formed by three subunits: troponin C (cTnC), 2 troponin I (cTnI), and troponin T (cTnT). The subunit cTnC is the Ca 2ϩ -binding protein, cTnI binds actin and inhibits actomyosin ATPase in relaxed muscle, and cTnT anchors the troponin complex on the actin filament. A prominent feature of cardiac muscle regulation is the Ca 2ϩ -dependent dynamic interactions among the thin filament proteins and the multiple structural transitions at the interface between troponin and the actin filament. These structural transitions include opening/closing of the N-domain of cTnC (3, 4), changes in conformation of both...

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation

“…This slightly higher concentration of actin ensures greater incorporation of troponin into functional regulatory units, but at the cost of cooperativity since troponin no longer occurs in every regulatory site. These same samples showed a significant increase in pCa 50 upon addition of S1-ADP, consistent with previous observations [13, 38]. …”

“…To implement FRET in this study, a series of recombinant single cysteine mutants were generated from wild type rat protein clones using approaches similar to those previously reported [10, 13, 37, 38]. Briefly, using the GeneTailor™ Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA), rat cDNA clones of wild-type cTnC, cTnI and cTnT sub-cloned into the plasmid pSBETa was used as a template DNA to generate single-cysteine cTnC and cTnI mutants and phosphomimetic mutants of cTnT.…”

Structural Dynamics of C-domain of Cardiac Troponin I Protein in Reconstituted Thin Filament

“…This suggests that transitions in the structural dynamics of the RR of cTnI are kinetically influenced by the IR from the N terminus and the MD from the C terminus and may hint at the RR becoming unstructured as it enters the Ca 2ϩ -free state. These observed transition kinetics are very close to the kinetics of Ca 2ϩ dissociation-induced structural changes of troponin acquired using FRET (40). Altogether, our stopped-flow results support the recently proposed fly casting model (13) and are consistent with the view that Ca 2ϩ dissociation induces a structural transition in an unstructured MD that promotes subsequent structural transitions in the RR and IR through "fly casting activity" (41) and that the transition of the RR seems to serve as a kinetic linkage between the transitions of the MD and the IR.…”

“…Based on our collective results, we propose a revised mechanistic scheme of Ca 2ϩ -induced thin filament deactivation as presented in Scheme 2. As in our previously proposed scheme (40), muscle relaxation is suggested to involve a "rapid-then-slow" two-step structural transition of the thin filament at the interface between cTn and actin. The fly casting activity of the MD of C-cTnI is responsible for initiating the rapid first step, and the second slow step involves the "from cTnC/to actin" switching activity of the IR of C-cTnI.…”

Structural kinetics of myosin by transient time-resolved FRET

“…Transient kinetics in myosin has been typically monitored by tryptophan fluorescence (6,7), but that signal provides no direct structural information. FRET does provide structural information, in the form of interprobe distance, and transient FRET experiments have provided a glimpse of structural kinetics in muscle and other proteins (8)(9)(10). However, previous transient FRET measurements have been limited to the monitoring of a single signal with continuous excitation and detection, whereas only nanosecond time-resolved FRET (TR-FRET), in response to pulsed excitation, can resolve multiple distances and quantitate disorder (11,12).…”