22 research outputs found

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

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    Cardiac thin filament deactivation is initiated by Ca 2+ 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

    Structural and kinetic effects of hypertrophic cardiomyopathy related mutations R146G/Q and R163W on the regulatory switching activity of rat cardiac troponin I

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    [Display omitted] ► FRET study of effects of HCM-related cTnI mutations in reconstituted thin filaments. ► R146G/Q and R163W each uniquely impact interactions between cTnI and cTnC or actin. ► Fly casting mechanism underlying deactivation preserved in spite of mutation. ► R146G/Q and R163W cause TF to enter pre relax like state in the absence of S1-ADP. ► In situ effects on force−Ca2+ relationship of myocardial fibers match in vitro. Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca2+ and myosin S1 dependent conformational transitions in rat cTn structure. Ca2+-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI in unique and various ways. However, kinetic rates of conformational transitions induced by Ca2+-dissociation were universally slowed when R146G/Q and R163W were present. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR−actin interaction were consistent with increased passive tension

    Structural and Kinetic Effects of PAK3 Phosphorylation Mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

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    Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca 2+, but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca 2+-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca 2+-induced changes in cTnI–cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC–cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca 2+ by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca 2+ dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI

    Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

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    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (∼5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50°C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB–RNase H scheme can be applied to a broad range of nucleic acid amplification methods

    Structural basis for the in situ Ca2+ sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges

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    The in situ structural coupling between the cardiac troponin (cTn) Ca 2+ -sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca 2+ titrations of cTnC(T13C/N51C) AEDENS/DDPM -reconstituted fibers showed that the Ca 2+ -dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force–Ca 2+ relationship. N-cTnC opening still occurred steeply during Ca 2+ titrations in the presence of 1 mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca 2+ -sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca 2+ levels, treatment with ADP•Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca 2+ titrations in the presence of ADP•Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca 2+ with increased Ca 2+ -sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca 2+ -sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI
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