In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation.

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-µm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.

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

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.

Structural basis for the in situ Ca(2+) sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges.

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 1mM 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.

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

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

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 Ca(2+) and myosin S1 dependent conformational transitions in rat cTn structure. Ca(2+)-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 Ca(2+)-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 dynamics of C-domain of cardiac troponin I protein in reconstituted thin filament.

The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca(2+) binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca(2+)-bound to the Ca(2+)-free dynamic states were significantly faster (53.2-116.8 s(-1)) than that of the IR (14.1 s(-1)). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca(2+) dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca(2+) signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.

Functionalization of micro- and nano-apertures with chromate-selective solvent polymeric membrane.

A new miniaturization approach to create micro- and nanoscale ion selective electrodes (ISEs) was demonstrated and the concept tested with an environmentally relevant chromate-selective membrane consisting of 7.7:62.2:31.1 wt % Aliquat336:2-NPOE:PVC. Apertures of 100 nM and 30 microM dimensions were made using MEMS fabrication techniques and functionalized through a macroscale application of solvent polymeric membrane. Performance studies for the microscale ISE showed a response slope of -58.6+/-5.6 mV decade(-1) and limit of detection (LOD) of 2.1 x 10(-5)+/-1.1 x 10(-5) M, versus -65.2+/-4.2 mV decade(-1) and 1.8 x 10(-5)+/-6 x 10(-6) M for the nanoscale ISE. This was consistent with control studies with carefully conditioned coated wire electrodes, which demonstrated a response slope of -61.7+/-2.4 mV decade(-1) and a LOD of 3.0 x 10(-6)+/-1 x 10(-6) M. Response times for the best micro- and nanoscale ISEs were in the 10-20 s timeframe. Electrical resistance measurements were in the GOmega range for the microscale ISEs and nanoscale ISEs. Appropriate ISE geometry was confirmed through AFM measurements and calculations based on electrical properties for micro- and nanoscale apertures. These micro- and nanoscale ISEs are expected to have significant impact in the field of microscale analytical processes.

Bilayer lipid membrane (BLM) based ion selective electrodes at the meso-, micro-, and nano-scales.

This paper presents a novel method for making micron-sized apertures with tapered sidewalls and nano-sized apertures. Their use in bilayer lipid membrane-based ion selective electrode design is demonstrated and compared to mesoscale bilayers and traditional PVC ion selective electrodes. Micron-sized apertures are fabricated in SU-8 photoresist films and vary in diameter from 10 to 40 microm. The tapered edges in SU-8 films are desired to enhance bilayer lipid membrane (BLM) formation and are fabricated by UV-light overexposure. Nano-apertures are made in boron diffused silicon film. The membranes are used as septa to separate two potassium chloride solutions of different concentrations. Lecithin BLMs are assembled on the apertures by ejecting lipid solution. Potassium ionophore, dibenzo-18-crown-6, is incorporated into BLMs by dissolving it in the lipid solution before membrane assembly. Voltage changes with increasing potassium ion concentrations are recorded with an A/D converter. Various ionophore concentrations in BLMs are investigated. At least a 1% concentration is needed for consistent slopes. Electrode response curves are linear over the 10(-6) to 0.1M range with a sub-Nernstian slope of 20mV per Log concentration change. This system shows high selectivity to potassium ions over potential interfering sodium ions. BLMs on the three different aperture sizes at the meso-, micro-, and nano-scales all show similar linear ranges and limits of detection (LODs) as PVC ion selective membranes.