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.

Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis.

This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms.

Isolation and characterization of a mesophilic Arthrospira maxima strain capable of producing docosahexaenoic acid.

A strain of the cyanobacterium Arthrospira was isolated from Lake Chahannaoer in northern China and was characterized according to microscopic morphology, photosynthetic oxygen-evolving activity, growth rate, and nutritional profile. Compared with thermophilic Arthrospira species occurring naturally in tropical and subtropical lakes, this isolate is mesophilic and grows optimally at ~20 degreesC. The total protein, fatty acid, phycocyanin, carotenoid, and chlorophyll a contents were 67.6, 6.1, 4.32, 0.29, and 0.76 grams per 100 grams of dry weight, respectively. The strain is rich in polyunsaturated fatty acids (PUFAs). An essential omega-3 fatty acid, docosahexaenoic acid (DHA), was detected, and gamma-linolenic acid (GLA) and DHA accounted for 28.3% of the total fatty acid content. These features of this newly isolated strain make it potentially useful in commercial mass culture in local areas or as a biofuel feedstock. It is also an alternative resource for studying the metabolic PUFA pathways and mechanisms of cold stress tolerance in cyanobacteria.

10,000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis.

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 µg mL⁻¹ and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL⁻¹. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.

Structural and kinetic effects of PAK3 phosphorylation mimic of cTnI(S151E) on the cTnC-cTnI interaction in the cardiac thin filament.

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.

Förster resonance energy transfer structural kinetic studies of cardiac thin filament deactivation.

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 Ca2+-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 Ca2+ titrations and stopped-flow kinetic measurements show that different thin filament structural transitions have different Ca2+ sensitivities and Ca2+ 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 Ca2+ 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 kinetics of cardiac troponin C mutants linked to familial hypertrophic and dilated cardiomyopathy in troponin complexes.

The key events in regulating cardiac muscle contraction involve Ca(2+) binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca(2+) movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca(2+) binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca(2+)-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca(2+). L29Q had no effect on the closing rate of the N-domain triggered by release of Ca(2+), but reduced the Ca(2+)-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca(2+) dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.

The two divergent PEP-carboxylase catalytic subunits in the green microalga Chlamydomonas reinhardtii respond reversibly to inorganic-N supply and co-exist in the high-molecular-mass, hetero-oligomeric Class-2 PEPC complex.

Our recent molecular studies revealed two divergent PEP-carboxylase (PEPC [Ppc]) encoding genes in the green microalga Chlamydomonas reinhardtii, CrPpc1 and CrPpc2, which are coordinately responsive to changes in inorganic-N and -C supply at the transcript level [Mamedov, T.G., Moellering, E.R. and Chollet, R. (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga C. reinhardtii, Plant J. 42, 832-843]. Here, we report the distribution of these two encoded catalytic subunits in the minor Class-1 and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, hetero-oligomeric complex containing both CrPpc1 (p109) and CrPpc2 (p131) polypeptides. The Class-1 enzyme, however, is a typical PEPC homotetramer comprised solely of p109. We also document that the amount of both CrPpc1/2 catalytic subunits is up-/down-regulated by varying levels of NH(4)(+) supplied to the culture medium.

The chloroplast Rieske iron-sulfur protein. At the crossroad of electron transport and signal transduction.

We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.

[Laboratory culture of Tetraselmis strains isolated from the coasts of China].

Tetraselmis is an important alga commonly used as live food in aquaculture. This paper reported the laboratory culture of its strains collected from the coasts of China, covering Guangdong, Zhejiang and Shandong Provinces. The separated strains of Tetraselmis for mono-species culture were isolated with capillary method, and their axenic cultures were obtained by inoculating them in plates with solid Marine III medium (M III). Modified Guillard & Ryther medium (MGM), M III, and modified Marine III medium (M III M) were utilized for the liquid cultivation of Tetraselmis in 500 mL flasks. It was displayed that the average growth of Tetraselmis cultured in M III M was the fastest at pH 7.0-8.0, and vitamin B12 addition to M III M slightly increased the relative growth rate of Tetraselmis. Therefore, the modified Marine III medium was effective to use as a large-scale culture medium for Tetraselmis in aquaculture.