Complex transformations of chemical signals passing through a passive barrier.

It has been recently observed that a passive barrier separating two excitable chemical media may transform the frequency of a train of incoming pulses. In this work we apply the FitzHugh-Nagumo-type model to study this phenomenon in a detailed way. Our numerical calculations demonstrate that at the barrier a periodic train of pulses may be transformed into a complex output signal. The ratio of frequencies of the output and the input signals, plotted as a function of the barrier's width or as a function of the input signal frequency, has a devil's-staircase-like shape.

Effect of 13-cis violaxanthin on organization of light harvesting complex II in monomolecular layers.

Lutein, neoxanthin and violaxanthin are the main xanthophyll pigment constituents of the largest light-harvesting pigment-protein complex of photosystem II (LHCII). High performance liquid chromatography analysis revealed photoisomerization of LHCII-bound violaxanthin from the conformation all-trans to the conformation 13-cis and 9-cis. Maximally, the conversion of 15% of all-trans violaxanthin to a cis form could be achieved owing to the light-driven reactions. The reactions were dark-reversible. The all-trans to cis isomerization was found to be driven by blue light, absorbed by chlorophylls and carotenoids, as well as by red light, absorbed exclusively by chlorophyll pigments. This suggests that the photoisomerization is a carotenoid triplet-sensitized reaction. The monomolecular layer technique was applied to study the effect of the 13-cis conformer of violaxanthin and its de-epoxidized form, zeaxanthin, on the organization of LHCII as compared to the all-trans stereoisomers. The specific molecular areas of LHCII in the two-component system composed of protein and exogenous 13-cis violaxanthin or 13-cis zeaxanthin show overadditivity, which is an indication of the xanthophyll-induced disassembly of the aggregated forms of the protein. Such an effect was not observed in the monomolecular layers of LHCII containing all-trans conformers of violaxanthin and zeaxanthin. 77 K chlorophyll a fluorescence emission spectra recorded from the Langmuir-Blodgett (L-B) films deposited to quartz from monomolecular layers formed with LHCII and LHCII in the two-component systems with all-trans and 13-cis isomers of violaxanthin and zeaxanthin revealed opposite effects of both conformers on the aggregation of the protein. The cis isomers of both xanthophylls were found to decrease the aggregation level of LHCII and the all-trans isomers increased the aggregation level. The calculated efficiency of excitation energy transfer to chlorophyll a from violaxanthin assumed to remain in two steric conformations was analyzed on the basis of the chlorophyll a fluorescence excitation spectra and the mean orientation of violaxanthin molecules in LHCII (71 degrees with respect to the normal to the membrane), determined recently in the linear dichroism experiments [Gruszecki et al., Biochim. Biophys. Acta 1412 (1999) 173-183]. The calculated efficiency of excitation energy transfer from the violaxanthin pool assumed to remain in conformation all-trans was found to be almost independent on the orientation angle within a variability range. In contrast the calculated efficiency of energy transfer from the form cis was found to be strongly dependent on the orientation and varied between 1.0 (at 67.48 degrees ) and 0 (at 70.89 degrees ). This is consistent with two essentially different, possible functions of the cis forms of violaxanthin: as a highly efficient excitation donor (and possibly energy transmitter between other chromophores) or purely as a LHCII structure modifier.

Xanthophyll pigments in light-harvesting complex II in monomolecular layers: localisation, energy transfer and orientation

Monomolecular layers of the largest light-harvesting pigment-protein complex of Photosystem II (LHCII) were formed at the argon-water interface. The molecular area of the LHCII monomer in monomolecular layers determined from the isotherms of compression is found to be close to 14 nm2, which corresponds well to the molecular dimensions of the protein evaluated on the basis of crystallographic studies. Monolayers of LHCII were deposited on a glass support by means of the Langmuir-Blodgett technique and subjected to spectroscopic studies: electronic absorption spectrophotometry and spectrofluorometry. The fluorescence excitation spectra of chlorophyll a in monolayers of LHCII were analysed using gaussian deconvolution. Comparison of the absorption and fluorescence excitation spectra enabled calculation of the rate of excitation energy transfer in the system. Excitation energy was found to be transferred to chlorophyll a from chlorophyll b with 97% efficiency, from neoxanthin with 85%, from lutein with 62% and from violaxanthin with at least 54% efficiency. The analysis of the position of the 0-0 absorption band of the xanthophylls revealed that neoxanthin is located in the same protein environment as lutein but in a different environment than violaxanthin. The analysis of fluorescence excitation spectra of chlorophyll a in LHCII, recorded with the excitation light beam polarised in two orthogonal directions, enabled the determination of the mean orientation angle of the accessory xanthophyll pigments with respect to the plane of the sample. The mean orientation of lutein found in this study (approx. 51 degrees ) corresponds well to the crystallographic data. Neoxanthin was found to adopt a similar orientation to lutein. The transition dipole moment of violaxanthin was found to form a mean angle of 71 degrees with the axis spanning two polar regions of the protein, perpendicular to the plane of the monolayer, suggesting planar orientation of this pigment with respect to the plane of the thylakoid membrane. These experimentally determined xanthophyll orientations are discussed in terms of importance of peripheral xanthophyll pigments in supramolecular organisation of LHCII and the operation of the xanthophyll cycle within the thylakoid membrane.

Spin-label studies on phosphatidylcholine-polar carotenoid membranes: effects of alkyl-chain length and unsaturation.

Spin-labeling methods were used to study the structure and dynamic properties of phosphatidylcholine (PC)-dihydroxycarotenoid membranes as a function of phospholipid alkyl chain length, alkyl chain saturation, temperature and mol fraction of carotenoids. (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin increase order and decrease motional freedom of the lipid alkyl chains in fluid-phase PC membranes. The effect of carotenoids decreases as the alkyl chain length of saturated PC increases. (2) The abrupt changes of spin-label motion observed at the main-phase transition of the saturated PC membranes are broadened and shifted to lower temperatures. At a carotenoid concentration of 10 mol%, they disappear for short-chain PC membranes (12-14 carbons), but are still observed for long-chain PC membranes (18-22 carbons). (3) In fluid-phase PC membranes possessing short alkyl chains (12-14 carbons), the activation energy of the rotational diffusion of 16-doxylstearic acid spin label (16-SASL) is significantly lower at a carotenoid concentration of 10 mol%. The difference decreases as the alkyl-chain length increases. (4) The presence of unsaturated alkyl chains greatly reduces the effects of carotenoids on the mobility of the polar headgroups as observed with tempocholine dipalmitoylphosphatidic acid ester and on the order of alkyl chains near the polar headgroup region as observed with 5-doxylstearic acid spin label (5-SASL). The effect of unsaturation is, however, moderate in the membrane center as shown with 16-SASL. Also, the effect of carotenoids on the order and motion of the rigid and highly anisotropic molecules dissolved in the PC membranes is significantly greater in saturated PC membranes.

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.

A simple model describing the kinetics of the xanthophyll cycle.

A new kinetic model of the xanthophyll cycle is proposed. The model is based on the assumption that the light-dependent interconversion of the so-called available and unavailable violaxanthin constitutes the rate-limiting process of the cycle at intermediate, non-saturating light intensities. This assumption, together with the known properties of violaxanthin de-epoxidase, explains all specific features of the experimental facts.

Galactolipid multibilayers modified with xanthophylls: orientational and diffractometric studies.

Oriented multibilayers of chloroplast galactolipids: monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) modified with violaxanthin and zeaxanthin were examined by X-ray diffractometry and linear dichroism. The results obtained suggest that zeaxanthin, in contrast to violaxanthin, has a significant ordering effect on galactolipid bilayers. The best ordered system consists of DGDG and zeaxanthin. In this case, the angle between the long axis of zeaxanthin molecule and the normal to the plane of bilayers amounts to 9 degrees and system has a periodicity of 61.7 A. The analogous angles in systems MGDG + violaxanthin, MGDG + zeaxanthin and DGDG + violaxanthin are clearly wider (35 degrees, 17 degrees and 28 degrees, respectively) but diffractograms show no distinct maxima.

Effect of polar carotenoids on the oxygen diffusion-concentration product in lipid bilayers. An EPR spin label study.

The oxygen diffusion-concentration product was determined in phosphatidylcholine (PC) bilayers from oxygen broadening of the spin label EPR spectra. The use of fatty acid spin labels makes it possible to do structural and oximetric measurements with the same sample. We find that polar carotenoids, zeaxanthin and violaxanthin, increase ordering of hydrocarbon chains in saturated (dimyristoyl-PC) and unsaturated (egg yolk PC) membranes and also significantly decrease the oxygen diffusion-concentration product in the hydrocarbon region of these membranes. At 25 degrees C in the presence of 10 mol% of carotenoids, the product is about 30% smaller than in pure PC membranes. Intercalation of carotenoids decreases the oxygen diffusion-concentration product in the central part of the bilayer and has little effect on the product in the polar head group region. In contrast, cholesterol molecules significantly reduce the product on and near the membrane surface, and do not change it (saturated PC) or increase it (unsaturated PC) in the middle of the bilayer (Subczynski, W.K., Hyde, J.S. and Kusumi, A. (1989) Proc. Natl. Acad. Sci. USA 86, 4474-4478). The decrease of oxygen diffusion-concentration product may be a mechanism of carotenoid protective activity, which should be effective in plant and animal cells in the light as well as in the dark.

Orientation of xanthophylls in phosphatidylcholine multibilayers.

Oriented multibilayers of dimyristoyl phosphatidylcholine (DMPC) modified with violaxanthin or zeaxanthin were examined by X-ray diffractometry and linear dichroism. It appears that pigment molecules and the normal to the bilayer plane form an angle of 24-25 degrees. It was also observed that rather small concentrations of added xanthophylls (molar fraction up to 3%) increase the pigmented bilayer thickness by a value of about 2 A as compared with that of the pure DMPC bilayer. The observed nonzero linear dichroism at normal incidence of light suggests the possibility of nonhomogeneous orientation of transition dipoles in the plane of the bilayer.