The effect of some antiseptic drugs on the energy transfer in chromatophore photosynthetic membranes of purple non-sulfur bacteria Rhodobacter sphaeroides.

Chromatophores of purple non-sulfur bacteria (PNSB) are invaginations of the cytoplasmic membrane that contain a relatively simple system of light-harvesting protein-pigment complexes, a photosynthetic reaction center (RC), a cytochrome complex, and ATP synthase, which transform light energy into the energy of synthesized ATP. The high content of negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) in PNSB chromatophore membranes makes these structures potential targets that bind cationic antiseptics. We used the methods of stationary and kinetic fluorescence spectroscopy to study the effect of some cationic antiseptics (chlorhexidine, picloxydine, miramistin, and octenidine at concentrations up to 100 µM) on the spectral and kinetic characteristics of the components of the photosynthetic apparatus of Rhodobacter sphaeroides chromatophores. Here we present the experimental data on the reduced efficiency of light energy conversion in the chromatophore membranes isolated from the photosynthetic bacterium Rb. sphaeroides in the presence of cationic antiseptics. The addition of antiseptics did not affect the energy transfer between the light-harvesting LH1 complex and reaction center (RC). However, it significantly reduced the efficiency of the interaction between the LH2 and LH1 complexes. The effect was maximal with 100 µM octenidine. It has been proved that molecules of cationic antiseptics, which apparently bind to the heads of negatively charged cardiolipin molecules located in the rings of light-harvesting pigments on the cytoplasmic surface of the chromatophores, can disturb the optimal conditions for efficient energy migration in chromatophore membranes.

The mitochondrial-targeted reactive species scavenger JP4-039 prevents sulfite-induced alterations in antioxidant defenses, energy transfer, and cell death signaling in striatum of rats.

Sulfite oxidase (SO) deficiency is a disorder caused either by isolated deficiency of SO or by defects in the synthesis of its molybdenum cofactor. It is characterized biochemically by tissue sulfite accumulation. Patients present with seizures, progressive neurological damage, and basal ganglia abnormalities, the pathogenesis of which is not fully established. Treatment is supportive and largely ineffective. To address the pathophysiology of sulfite toxicity, we examined the effects of intrastriatal administration of sulfite in rats on antioxidant defenses, energy transfer, and mitogen-activated protein kinases (MAPK) and apoptosis pathways in rat striatum. Sulfite administration decreased glutathione (GSH) concentration and glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and glutathione reductase activities in striatal tissue. Creatine kinase (CK) activity, a crucial enzyme for cell energy transfer, was also decreased by sulfite. Superoxide dismutase-1 (SOD1) and catalase (CAT) proteins were increased, while heme oxygenase-1 (HO-1) was decreased. Additionally, sulfite altered phosphorylation of MAPK by decreasing of p38 and increasing of ERK. Sulfite further augmented the content of GSK-3ß, Bok, and cleaved caspase-3, indicating increased apoptosis. JP4-039 is a mitochondrial-targeted antioxidant that reaches higher intramitochondrial levels than other traditional antioxidants. Intraperitoneal injection of JP4-039 before sulfite administration preserved activity of antioxidant enzymes and CK. It also prevented or attenuated alterations in SOD1, CAT, and HO-1 protein content, as well as changes in p38, ERK, and apoptosis markers. In sum, oxidative stress and apoptosis induced by sulfite injection are prevented by JP4-039, identifying this molecule as a promising candidate for pharmacological treatment of SO-deficient patients.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

Fluorescent Property of Chitosan Oligomer and Its Application as a Metal Ion Sensor.

An aqueous solution was successfully prepared using a low-molecular-weight chitosan oligomer and FITC, and its structural and fluorescent properties were observed by using ¹H NMR, 13C NMR, FT-IR, XRD, UV-Vis, and PL spectrometry. Its application as a metal ion sensor was also evaluated. The fluorescence in the water-soluble chitosan oligomer was a result of the carbamato anion (NHCOO-), and a synthesized FITC-labeled chitosan oligomer exhibited an effective detection effect for copper ion as well as energy transfer by the ion near FITC that caused a fluorescence decrease (quenching). The chitosan oligomer was confirmed to be applicable as a selective and sensitive colorimetric sensor to detect Cu2+.

Caffeine ingestion improves power output decrement during 3-min all-out exercise.

PURPOSE: To investigate the effect of caffeine ingestion on the 3-min all-out test (3MT) performance and plasma electrolytes in athletes. METHODS: Fifteen collegiate male basketball players were recruited and completed two trials separated by at least 1 week in caffeine (CAF, 6 mg kg(-1)) and placebo conditions. During the first visit, participants performed an incremental cycling test to determine their 3MT resistance. After a familiarization trial, participants performed a CAF or PL trial according to a randomized crossover design. One hour after ingesting capsules, the participants performed the 3MT to estimate the end-test power (EP) and work done above EP (WEP). Blood samples for sodium (Na(+)), potassium (K(+)), pH, and lactate concentrations were drawn pretest, 1 h after ingestion, and posttest. RESULTS: Significant differences in WEP (CAF vs. PL, 13.4 ± 3.0 vs. 12.1 ± 2.7 kJ, P < 0.05) but not in EP (CAF vs. PL, 242 ± 37 vs. 244 ± 42 W, P > 0.05) were determined between the conditions. Compared with the PL condition, the CAF condition yielded significantly higher power outputs (60-150 s), a lower fatigue rate during the 3MT (CAF vs. PL, 0.024 ± 0.007 vs. 0.029 ± 0.006 s(-1), P < 0.05), a significantly higher lactate concentration after the 3MT, and significantly lower K(+) concentrations at 1 h after caffeine ingestion. There were no significant interaction effects for pH and Na(+) concentrations. CONCLUSIONS: Caffeine ingestion did not change EP but improved WEP and the rate of decline in power output during short-term, severe exercise.

Energy-dissipative supercomplex of photosystem II associated with LHCSR3 in Chlamydomonas reinhardtii.

Plants and green algae have a low pH-inducible mechanism in photosystem II (PSII) that dissipates excess light energy, measured as the nonphotochemical quenching of chlorophyll fluorescence (qE). Recently, nonphotochemical quenching 4 (npq4), a mutant strain of the green alga Chlamydomonas reinhardtii that is qE-deficient and lacks the light-harvesting complex stress-related protein 3 (LHCSR3), was reported [Peers G, et al. (2009) Nature 462(7272):518-521]. Here, applying a newly established procedure, we isolated the PSII supercomplex and its associated light-harvesting proteins from both WT C. reinhardtii and the npq4 mutant grown in either low light (LL) or high light (HL). LHCSR3 was present in the PSII supercomplex from the HL-grown WT, but not in the supercomplex from the LL-grown WT or mutant. The purified PSII supercomplex containing LHCSR3 exhibited a normal fluorescence lifetime at a neutral pH (7.5) by single-photon counting analysis, but a significantly shorter lifetime at pH 5.5, which mimics the acidified lumen of the thylakoid membranes in HL-exposed chloroplasts. The switch from light-harvesting mode to energy-dissipating mode observed in the LHCSR3-containing PSII supercomplex was sensitive to dicyclohexylcarbodiimide, a protein-modifying agent specific to protonatable amino acid residues. We conclude that the PSII-LHCII-LHCSR3 supercomplex formed in the HL-grown C. reinhardtii cells is capable of energy dissipation on protonation of LHCSR3.

Differences in energy transfer of a cyanobacterium, Synechococcus sp. PCC 7002, grown in different cultivation media.

Currently, cyanobacteria are regarded as potential biofuel sources. Large-scale cultivation of cyanobacteria in seawater is of particular interest because seawater is a low-cost medium. In the present study, we examined differences in light-harvesting and energy transfer processes in the cyanobacterium Synechococcus sp. PCC 7002 grown in different cultivation media, namely modified A medium (the optimal growth medium for Synechococcus sp. PCC 7002) and f/2 (a seawater medium). The concentrations of nitrate and phosphate ions were varied in both media. Higher nitrate ion and/or phosphate ion concentrations yielded high relative content of phycobilisome. The cultivation medium influenced the energy transfers within phycobilisome, from phycobilisome to photosystems, within photosystem II, and from photosystem II to photosystem I. We suggest that the medium also affects charge recombination at the photosystem II reaction center and formation of a chlorophyll-containing complex.

Roles of the cyclic electron flow around PSI (CEF-PSI) and O2-dependent alternative pathways in regulation of the photosynthetic electron flow in short-term fluctuating light in Arabidopsis thaliana.

To assess the roles of the cyclic electron flow around PSI (CEF-PSI) and O2-dependent alternative pathways including the water-water cycle in fluctuating light, we grew the wild type and pgr5 mutant of Arabidopsis thaliana in constant light, and measured Chl fluorescence and P700 parameters in their leaves in the fluctuating light alternating between 240 (HL) and 30 µmol photons m⁻² s⁻² (LL) every 2 min. At 20% O2, the photochemical quantum yield of PSII decreased, in particular in the pgr5 plants, soon after the start of the fluctuating light treatment. PSI of the pgr5 plants was markedly photoinhibited by this treatment for 42 min. Slight PSI photoinhibition was also observed in the wild type. We measured energy sharing between PSII and PSI and estimated the PSI and PSII electron transport rates (ETRs). pgr5 showed larger energy allocation to PSI. In contrast to the wild type, the ratio of the PSI to the PSII ETR in pgr5 was higher in LL but lower in HL at 20% O2 due to PSI acceptor-side limitation. At 2.7% or 0% O2, the CEF-PSI of the pgr5 plants was enhanced, the acceptor-side limitation of PSI electron flow was released and PSI photoinhibition was not observed. The results suggest that the light fluctuation is a potent stress to PSI and that the CEF-PSI is essential to protect PSI from this stress.

Porphyra-334 isolated from the marine algae Bangia atropurpurea: conformational performance for energy conversion.

Prophyra-334 (p-334) may play a role of energy transfer under an uncertain mechanism, and we speculate the possible model. Via 1D and 2D NMR experiments, it was simulated the correlation between dissociation and conformation of p-334. Intramolecular interactions were observed based on a series of changes in the 1H and 13C chemical shifts. Nuclear Overhauser effect spectroscopy experiments and molecular models in various pD conditions indicated the p-334 molecular dissociation process status. In addition, we also used Chem3D software to find the most possible molecular conformation. The relationship between the structural status and energy conversion is explained. Those are the primary results. More researches on it are highly expected in the future.

Linear rate-equilibrium relations arising from ion channel-bilayer energetic coupling.

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the single-molecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.

Stabilization and modulation of the phycobilisome by calcium in the calciphilic freshwater red alga Bangia atropurpurea.

The bangiophycean filamentous red alga Bangia atropurpurea is distributed in freshwater habitats such as littoral and splash zones of lakes or rapid currents distant from the sea. In these habitats, the distribution and growth of this alga appear to be related to hard water rich in calcium ions. To characterize the eco-physiological properties of this calciphilic red alga, we examined the effects of long-term and short-term Ca(2+) depletion on photosynthetic growth of the thallus and on the phycobilisome. Long-term culture experiments suggested that higher Ca(2+) concentrations (>50mgL(-1)) were required to sustain thallus growth and pigmentation of cells. In short-term Ca(2+)-depletion treatments, fluorescence derived from phycoerythrin (PE) fluctuated, although the absorption spectra of the thalli did not change. After 30 min of Ca(2+) depletion, the fluorescence lifetime of PE became markedly longer, indicating that the energy transfer from PE to phycocyanin (PC) was suppressed. The fluorescence lifetime of PE returned to its original value within a short time after 4h of Ca(2+) depletion, however, energy transfer from PE to PC was still suppressed. This suggested that the excitation energy absorbed by PE was quenched during prolonged Ca(2+) depletion. The efficient energy transfer from PC and allophycocyanin were unchanged during these treatments.

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain.

A reversible structural unlocking reaction, in which the close-packed van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (DeltaH(0) = 35 +/- 4 kJ/mol) which is nearly compensated in free energy by the entropy change (DeltaS(0) = 112 +/- 20 Jxmol(-1)xK(-1)). The unlocking reaction has a time constant of about 1 mus at 5 degrees C and is enthalpy-limited with an activation energy of 32 +/- 1 kJ/mol and a large Arrhenius preexponential factor of A = 7.5 x 10(11) s(-1). In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 +/- 1 kJ/mol leads to unfolding of the C-terminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the C-terminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.

Conjugated polymers for light-activated antifungal activity.

A cationic polythiophene-porphyrin (PTP) dyad is shown to exhibit efficient light-activated antifungal activity. Higher singlet oxygen (¹O2) generation efficiency can be attained from PTP upon photoexcitation due to the light-harvesting properties of the polymer backbone and efficient energy transfer from the polythiophene to the porphyrin units. PTP can be used for treating fungal infections in lower doses of irradiation light and polymer concentration.

Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems.

Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems' activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30-40% for both photoreactions-P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core-membrane linker allophycocyanin APC-L(CM) is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-L(CM) may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.

A novel and mild isolation procedure of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum.

In this article, we developed a new and mild procedure for the isolation of chlorosomes from a green sulfur bacterium Chlorobaculum tepidum. In this procedure, Fenna-Matthews-Olson (FMO) protein was released by long cold treatment (6°C) of cells under the presence of a chaotrope (2 M NaSCN) and 0.6 M sucrose. Chlorosomes were released by an osmotic shock of the cold-treated cells after the formation of spheroplasts without mechanical disruption. Chlorosomes were finally purified by a sucrose step-wise density gradient centrifugation. We obtained two samples with different density (20 and 23% sucrose band, respectively) and compared them by SDS-PAGE, absorption spectroscopy at 80 K, fluorescence and CD spectroscopy at room temperature. Cells whose absorption maximum was longer than 750 nm yielded higher amount of the 20% sucrose fraction than those having an absorption maximum shorter than 750 nm.

Single-molecule imaging of EGFR signalling on the surface of living cells.

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Evaluation of binding and thermodynamic characteristics of interactions between a citrus flavonoid hesperitin with protein and effects of metal ions on binding.

The mechanism of interaction of a non-glycosidic citrus flavonoid, hesperitin (HES) with bovine serum albumin (BSA) was studied by UV-vis absorption, fluorescence, FT-IR, circular dichroism, fluorescence anisotropy and synchronous fluorescence spectroscopy in phosphate buffer of pH 7.4. Fluorescence data revealed that the fluorescence quenching of BSA by HES was the result of the formed complex of HES-BSA. The binding constants and thermodynamic parameters at four different temperatures, the location of binding, and the nature of binding force were determined. The hydrogen bonds interactions were found to be the predominant intermolecular forces to stabilize the complex. The conformation of BSA was discussed by synchronous fluorescence and CD methods. The alterations of protein secondary structure upon complexation with HES were evident from the gradual decrease in α-helicity. The distance between the donor (BSA) and acceptor (flavonoid) was calculated from the fluorescence resonance energy transfer and found to be 1.978 nm. Common ions viz., Zn(2+), K(+), Cu(2+), Ni(2+), Mn(2+) and Co(2+) were found to influence the binding of flavonoid to protein.

Regulatory subunit I-controlled protein kinase A activity is required for apical bile canalicular lumen development in hepatocytes.

Signaling via cAMP plays an important role in apical cell surface dynamics in epithelial cells. In hepatocytes, elevated levels of cAMP as well as extracellular oncostatin M stimulate apical lumen development in a manner that depends on protein kinase A (PKA) activity. However, neither the identity of PKA isoforms involved nor the mechanisms of the cross-talk between oncostatin M and cAMP/PKA signaling pathways have been elucidated. Here we demonstrate that oncostatin M and PKA signaling converge at the level of the PKA holoenzyme downstream of oncostatin M-stimulated MAPK activation. Experiments were performed with chemically modified cAMP analogues that preferentially target regulatory subunit (R) I or RII holoenzymes, respectively, in hepatocytes. The data suggest that the dissociation of RI- but not RII-containing holoenzymes, as well as catalytic activity of PKA, is required for apical lumen development in response to elevated levels of cAMP and oncostatin M. However, oncostatin M signaling does not stimulate PKA holoenzyme dissociation in living cells. Based on pharmacological and cell biological studies, it is concluded that RI-controlled PKA activity is essential for cAMP- and oncostatin M-stimulated development of apical bile canalicular lumens.

Spectroscopic Investigation of DCCH and FTSC as a potential pair for Förster Resonance Energy Transfer in different solvents.

Two molecules, 7-(diethylamino)coumarin-3-carbohydrazide (DCCH) and fluorescein-5-thiosemicarbazide (FTSC) were investigated in different solvents, under varying pH conditions regarding their spectroscopic properties for the usage as a Förster Resonance Energy Transfer (FRET) pair to study the molecular interaction between cellulosic surfaces. All the relevant spectroscopic properties to determine the Förster distance were measured and the performance as a FRET system was checked. From the results, it is clear that the environmental conditions need to be accurately controlled as both, but especially the FTSC dyes are sensitive to changes. For high enough concentrations positive FRET systems were observed in DMF, DMSO, H2O, THF and alkaline DMF. However due to the low quantum yield of the unmodified DCCH throughout the investigated parameter range and the strong environmental dependency of FTSC, both dyes are not preferable for being used in a FRET system for studying interaction between cellulosic surfaces.