Isolation of Lamellar Aggregates of the Light-Harvesting Chlorophyll a/b Protein Complex of Photosystem II with Long-Range Chiral Order and Structural Flexibility

Simidjiev, Ilian; Barzda, Virginijus; Mustárdy, László; Garab, Győző

doi:10.1006/abio.1997.2204

Cited by 72 publications

(60 citation statements)

References 23 publications

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“…LHCII macroaggregates were isolated as described earlier from leaves of 2-wk-old pea (Pisum sativum, L.) grown in the greenhouse (10). Two types of preparations were used.…”

Section: Methodsmentioning

confidence: 99%

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteinsin vitro

Simidjiev

Stoylova

Amenitsch

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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144

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In many biological membranes, the major lipids are ''non-bilayer lipids,'' which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that the in vitro association of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a͞b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.circular dichroism ͉ chloroplast thylakoid membranes ͉ electron microscopy ͉ light-harvesting complex ͉ lipid-protein interactions T he lamellar organization of biological membranes provides a structural matrix for various proteins and controls the permeability of organic molecules, water, and ions; it also prevents nonspecific protein-protein aggregation, whereas it allows protein diffusion and conformational changes in the membrane. However, biomembranes usually contain substantial amounts of non-bilayer lipids, which in purified form assume nonlamellar structures. In fact, in many membranes, e.g., thylakoid membranes of chloroplasts, membranes of Escherichia coli, rhodopsin, and mitochondria, non-bilayer lipids constitute about half or more of the total lipid content. It is well established that the physical and functional properties of these membranes depend to a large extent on protein-lipid interactions (1, 2). There are a few examples showing that lipid polymorphism can be modulated by proteins, and, in some cases, small unilamellar vesicles can be reconstituted from non-bilayer lipids and membrane proteins (e.g., refs. 3 and 4). However, the structural role of large amounts of non-bilayer lipids has remained enigmatic, and the assembly of extended bilayer lamellae from proteins and predominantly non-bilayer lipids is poorly understood (1, 2, 5). In this work, we use a simple system, the two main constituents of pea thylakoid membranes, purified non-bilayer lipids and isolated protein complexes, to demonstrate that the formation of a large, ordered lamellar structure is possible even in the presence of large amounts of lipids.In chloroplast thylakoid membranes of g...

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“…LHCII macroaggregates were isolated as described earlier from leaves of 2-wk-old pea (Pisum sativum, L.) grown in the greenhouse (10). Two types of preparations were used.…”

Section: Methodsmentioning

confidence: 99%

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteinsin vitro

Simidjiev

Stoylova

Amenitsch

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

144

View full text Add to dashboard Cite

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“…All procedures were carried at 0°C to 4°C. PSII core particles and LHCII were isolated according to Ghanotakis et al (1984) and Simidjiev et al (1997), respectively. The isolated complexes were stored in 0.5-mL Eppendorf tubes under 280°C.…”

Section: Isolation Of Thylakoid Membranes Psii Core Particles and Lmentioning

confidence: 99%

Heat Stress Induces an Aggregation of the Light-Harvesting Complex of Photosystem II in Spinach Plants

et al. 2006

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Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25°C-50°C) in the dark for 30 min. At temperatures higher than 35°C, CO 2 assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45°C and decreased only slightly at 50°C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25°C to 50°C in the dark for 10 min. At temperatures over 35°C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25°C to 50°C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35°C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.

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“…Subchloroplast particles, fragments containing a few grana, were obtained by sonication and differential centrifugation (Jacobi 1971); they were resuspended in the same reaction medium. Lamellar aggregates of LHCII were obtained as described earlier (Simidjiev et al 1997) and suspended in 10 mM Tricine buffer (pH 7.6). Intact thylakoid membranes were oriented in a homogenous magnetic field of 1.5 T, and trapped in the oriented state by inclusion in a polyacrylamide gel (Finzi et al 1989).…”

Section: Methodsmentioning

confidence: 99%

Alignment of biological microparticles by a polarized laser beam

et al. 2005

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The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581-1590 have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic lightharvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.

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Isolation of Lamellar Aggregates of the Light-Harvesting Chlorophyll a/b Protein Complex of Photosystem II with Long-Range Chiral Order and Structural Flexibility

Cited by 72 publications

References 23 publications

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteinsin vitro

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteinsin vitro

Heat Stress Induces an Aggregation of the Light-Harvesting Complex of Photosystem II in Spinach Plants

Alignment of biological microparticles by a polarized laser beam

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