Zeaxanthin (dihydroxy-β-carotene) but not β-carotene rigidifies lipid membranes: a 1H-NMR study of carotenoid-egg phosphatidylcholine liposomes

Abstract1H-NMR technique was applied to study liposomes formed with egg-yolk phosphatidylcholine containing as an additional component two carotenoid pigments: β-carotene or zeaxanthin (dihydroxy-β-carotene). A strong rigidifying effect of zeaxanthin but not of β-carotene with respect to hydrophobic core of lipid bilayer was concluded from the carotenoid-dependent broadening of the NMR lines assigned to -CH2- groups and terminal -CH3 groups of lipid alkyl chains. A similar effect of zeaxanthin with respect to polar headgroups was concluded on the basis of the effect of the pigment on the shape of NMR lines attributed to -N+(CH3)3 groups. In contrast, β-carotene increases motional freedom of lipid polar headgroups. The inclusion of both carotenoids to liposomes resulted in the enhanced penetration of Pr3+ ions to the polar zone of the external layer of a membrane monitored by the splitting of the -N+(CH3)3 signal, the effect of β-carotene being much more pronounced. Differences in the effect on membrane structure and molecular dynamics observed for β-carotene and its polar derivative are discussed in terms of organization of a carotenoid-containing lipid membrane

Interaction of ferredoxin:NADP+ oxidoreductase with model membranes

AbstractThe ferredoxin:NADP+ oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR–membrane interaction is discussed

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

AbstractPolar carotenoid pigment zeaxanthin (β,β-carotene-3,3′-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13×107 Ω cm2 (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H+-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV–Vis linear dichroism analysis of multibilayer formed with the same lipid–carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21° with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of β-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane

AbstractLysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon–water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% β-sheet, 28% aggregated β-strands, 10% α-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of α-helices, turns and loops, and β-structures did not change, however, the 1636cm−1 β-sheet band increased from 18% to 31% at the expense of the 1680cm−1 β-sheet structure. Spectral analysis of the amide I band showed that the α-helical component was oriented with at 41° to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP+ oxidoreductase

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP(+) oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed. Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users

Zeaxanthin epoxidation : an in vitro approach

Zeaxanthin epoxidase (ZE) is an enzyme operating in the violaxanthin cycle, which is involved in photoprotective mechanisms. In this work model systems to study zeaxanthin (Zx) epoxidation were developed. Two assay systems are presented in which epoxidation of Zx was observed. In these assays two mutants of Arabidopsis thaliana which have active only one of the two xanthophyll cycle enzymes were used. The npq1 mutant possesses an active ZE and is thus able to convert Zx to violaxanthin (Vx) but the violaxanthin de-epoxidase (VDE) is inactive, so that Vx cannot be converted to Zx. The other mutant, npq2, possesses an active VDE and can convert exogenous Vx to Zx under strong light conditions but reverse reaction is not possible. The first assay containing thylakoids from npq1 and npq2 mutants of A. thaliana gave positive results and high efficiency of epoxidation reaction was observed. The amount of Zx was reduced by 25%. To optimize high efficiency of epoxidation reaction additional factors facilitating both fusion of the two types of thylakoids and incorporation of Zx to their membranes were also studied. The second kind of assay contained npq1 mutant thylakoids of A. thaliana supplemented with exogenous Zx and monogalactosyldiacylglycerol (MGDG). Experiments with different proportions of Zx and MGDG showed that their optimal ratio is 1:60. In such system, due to epoxidation, the amount of Zx was reduced by 38% of its initial level. The in vitro systems of Zx epoxidation described in this paper enable analysis some properties of the ZE without necessity of its isolation