Effects of a non-ionic detergent on the spectral properties and aggregation state of the light-harvesting chlorophyll a/b protein complex (LHCII)

Bassi, Roberto; Silvestri, Mariuccia; Dainese, Paola; Moya, I.; Giacometti, Giorgio M.

doi:10.1016/1011-1344(91)80170-m

Cited by 55 publications

(33 citation statements)

References 34 publications

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“…In the case of LHCII, 92-95% of the total emission was due to a 3.8 ns lifetime component (T3) and the rest (a few percent) to a 1.4 ns component (T2). These values are in excellent agreement with the results of previous work with LHCII (41,42). The emission spectrum of the elementary components has been obtained by deconvolution of the decays measured at different wavelengths (see Materials and Methods).…”

Section: Purification and Biochemical Characterization Of Lhcbsupporting

confidence: 80%

“…It is known that the spectral properties and fluorescence decay behavior of pigment-protein complexes depend on interactions with the environment and, particularly, on their aggregation state (41,42). From this point of view, Chl-proteins isolated in detergent micelles represent an artificial system, which easily allows investigation of properties due to specific antenna subunits but in a situation rather far from physiological, i.e., where chlorophyll-protein interactions with each other and with thylakoid membrane lipids are not reproduced.…”

Section: Purification and Biochemical Characterization Of Lhcbmentioning

confidence: 99%

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Time-Resolved Fluorescence Analysis of the Photosystem II Antenna Proteins in Detergent Micelles and Liposomes

et al. 2001

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We have studied the time-resolved fluorescence properties of the light-harvesting complexes (Lhc) of photosystem II (Lhcb) in order to obtain information on the mechanism of energy dissipation (non-photochemical quenching) which is correlated to the conversion of violaxanthin to zeaxanthin in excess light conditions. The chlorophyll fluorescence decay of Lhcb proteins LHCII, CP29, CP26, and CP24 in detergent solution is mostly determined by two lifetime components of 1.2-1.5 and 3.6-4 ns while the contribution of the faster component is higher in CP29, CP26, and CP24 with respect to LHCII. The xanthophyll composition of Lhc proteins affects the ratio of the lifetime components: when zeaxanthin is bound into the site L2 of LHCII, the relative amplitude of the faster component is increased and, consequently, the chlorophyll fluorescence quenching is enhanced. Analysis of quenching in mutants of Arabidopsis thaliana, which incorporate either violaxanthin or zeaxanthin in their Lhc proteins, shows that the extent of quenching is enhanced in the presence of zeaxanthin. The origin of the two fluorescence lifetimes was analyzed by their temperature dependence: since lifetime heterogeneity was not affected by cooling to 77 K, it is concluded that each lifetime component corresponds to a distinct conformation of the Lhc proteins. Upon incorporation of Lhc proteins into liposomes, a quenching of chlorophyll fluorescence was observed due to shortening of all their lifetime components: this indicates that the equilibrium between the two conformations of Lhcb proteins is displaced toward the quenched conformation in lipid membranes or thylakoids with respect to detergent solution. By increasing the protein density in the liposomes, and therefore the probability of protein-protein interactions, a further decrease of fluorescence lifetimes takes place down to values typical of quenched leaves. We conclude that at least two major factors determine the quenching of chlorophyll fluorescence in Lhcb proteins, i.e., intrasubunit conformational change and intersubunit interactions within the lipid membranes, and that these processes are both important in the photoprotection mechanism of nonphotochemical quenching in vivo.The photosynthetic apparatus of higher plants is composed of two photosystems (PS), 1 PSI and PSII, each consisting of a core complex moiety, binding chlorophyll (Chl) a and -carotene, and a peripheral antenna system, binding Chl a, Chl b, and xanthophylls (Cars). The major component of the PSII antenna system is LHCII, a heterotrimer of the Lhcb1, -2, and -3 gene products (also called LHCIIb) binding half of the thylakoid pigments. Minor components are CP29 (LHCIIa), CP26 (LHCIIc), and CP24 (LHCIId), respectively the products of the Lhcb4, -5, and -6 genes, which are monomeric and connect the PSII core complex to the peripheral LHCII trimers.The large absorption cross-section of the antenna systems allows plants to grow in dim light. However, the light intensity experienced by a plant varies largely over ...

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Section: Purification and Biochemical Characterization Of Lhcbsupporting

confidence: 80%

Section: Purification and Biochemical Characterization Of Lhcbmentioning

confidence: 99%

Time-Resolved Fluorescence Analysis of the Photosystem II Antenna Proteins in Detergent Micelles and Liposomes

et al. 2001

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“…Clear differences in peak shape are observed in the Soret region. The typical CD spectrum of the LHCII control shows a major negative signal at 491 nm and a shoulder at 474 nm, in agreement with a previous report on LHCII in the monomeric state (25). In the case of the complex reconstituted with lutein only (LHCII lutein), the relative amplitude of these two peaks is reversed, with the 474 nm (Ϫ)-signal becoming predominant.…”

Section: Spectrasupporting

confidence: 75%

Carotenoid-binding Sites of the Major Light-harvesting Complex II of Higher Plants

Croce

Weiß

Bassi

1999

Journal of Biological Chemistry

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237

273

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Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in 1 O 2 * scavenging.Light energy for the photosynthesis of green plants is collected by an antenna system composed of many homologous proteins belonging to the Lhc multigene family (1). These pigment-protein complexes are organized around photosynthetic reaction centers to form supramolecular complexes embedded into the thylakoid membrane, accounting for ϳ70% of the pigment involved in plant photosynthesis. LHCII 1 is the most abundant light-harvesting complex in higher plants. The structure of this complex has been resolved at 3.4 Å by electron microscopy (2) and is formed by three hydrophobic transmembrane helices connected by hydrophilic loops and an amphipathic helix exposed to the luminal surface of the membrane. LHCII coordinates 7 Chl a, 5 Chl b, and 3-4 carotenoid molecules (lutein, neoxanthin, and a substoichiometric amount of violaxanthin) depending on the genotype (3) and the physiological state of the plant (4). In the structural model of LHCII (2), 2 xanthophyll molecules have been located in the center of the complex, forming an internal cross-brace interacting with helices A and B. These appear to be crucial for protein stabilization, as suggested by the fact that a stable LHCII complex cannot be obtained without lutein in refolding experiments (5, 6). Although the 2 central molecules were tentatively assigned to lutein (2), the structural resolution is insufficient for their identification and for the location of the xanthophyll molecule with respect to the 2 detected by structural analysis. The nature and location of the binding site for the third xanthophyll molecule are presently unknown. It is also unclear if the individual binding sites have different affinities for the three xanthophyll species.Carotenoids have at least five different roles in photosynthesis: 1) light h...

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“…3) as previously shown for the native complex purified from thylakoids (27,28). This protein could be made into trimers by incubation with lipid extract leading to the changes on absorption and CD spectra previously reported to be due to trimerization in native LHCII (27,28). Here we report on monomeric proteins.…”

Section: Resultsmentioning

confidence: 92%

Chlorophyll Binding to Monomeric Light-harvesting Complex

Remelli

Varotto

Sandonà

et al. 1999

Journal of Biological Chemistry

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208

199

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The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q y transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.In green plants light energy for photosynthesis is collected by an antenna system, made of many homologous proteins belonging to the Lhc multigene family (1). These pigment proteins are organized around photosynthetic reaction centers to form supramolecular complexes embedded into the thylakoid membranes. Lhc proteins bind about 70% of the pigments involved in plant photosynthesis. Understanding of energy transfer processes in the antenna and reaction centers requires recognition of the topological organization of subunits (2-4) and knowledge of three major parameters, namely: (i) the distances between chromophores; (ii) the mutual orientation of dipole transition moments; and (iii) the absorption/fluorescence energy levels. Although the elucidation of LHCII structure at 3.4 Å resolution (5) has allowed localization of chlorophyll-binding sites and of their relative distances, identification of transition dipole orientation and energy levels are precluded by insufficient resolution of the structure so far obtained or are not accessible by structural methods. Among Lhc proteins the most abundant is LHCII, which can be isolated as an heterotrimeric complex of the Lhcb1-3 gene products (6). LHCII binds 7 Chl 1 a, 5 Chl b, and three xanthophyll molecules/mol of polypeptide (1.6 -1.8 mol of lutein, 0.2-0.4 mol of violaxanthin, and 1.0 mol of neoxanthin) and is the best characterized Lhc polypeptide. (5, 7-10). Knowledge of the energy transfer factors for this protein would be a major step toward elucidation of light harvesting function. In this study we have used mutation analysis with the aim of the identification and characterization of the chromophores bound to each site; a series of mutant apoproteins was constructed by overexpression in bacteria ...

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Effects of a non-ionic detergent on the spectral properties and aggregation state of the light-harvesting chlorophyll a/b protein complex (LHCII)

Cited by 55 publications

References 34 publications

Time-Resolved Fluorescence Analysis of the Photosystem II Antenna Proteins in Detergent Micelles and Liposomes

Time-Resolved Fluorescence Analysis of the Photosystem II Antenna Proteins in Detergent Micelles and Liposomes

Carotenoid-binding Sites of the Major Light-harvesting Complex II of Higher Plants

Chlorophyll Binding to Monomeric Light-harvesting Complex

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