Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.
the role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content-yet their functional state is a bilayer. in vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31 p-nMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH-and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. these reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase. The primary function of biological membranes is to allow compartmentalization of cells and cellular organelles and, in general, the separation of two aqueous phases with different compositions. The functioning of these membranes, at the basic level, depends on the organization of their lipid molecules into bilayer structures 1-3. These structures provide a two-dimensional matrix, which is capable of embedding intrinsic proteins and permits the lateral diffusion of mobile compounds inside the 2D matrix of the membrane. By acting as highly selective barrier, the bilayer membrane allows the formation of concentration gradients of ions and other water-soluble compounds across them. The generation and utilization of the transmembrane electrochemical potential gradient for protons, Δµ H + or proton-motive force, is of pivotal importance in biological energy conversion 4. Most membrane lipids readily form bilayers. However, biological membranes also contain non-bilayer lipid species-which do not self-assemble into bilayers 5. Their role in the biomembranes is still enigmatic. Most noteworthy, in energy-converting membranes non-bilayer lipids constitute about half of their total lipid content,
In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment-pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while "psi-type" CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.
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