Reconstitution of Light Harvesting Complexes: A Single Apoprotein Binds CHLa, CHLb, and Xanthophylls

Cammarata, Kirk V.; Plumley, F. Gerald; Schmidt, Gregory W.

doi:10.1007/978-94-009-0511-5_302

Cited by 7 publications

(13 citation statements)

References 10 publications

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“…3. A single L H C I I gene product has binding sites for Chls a and b and xanthophylls (Cammarata et al 1990, Paulsen et al 1990, Cammarata and Schmidt 1992. 4.…”

Section: Characterization Of the Pigments And Proteins Which Reconstimentioning

confidence: 99%

“…The pigment-binding properties of the products of individual LHC II and LHC I genes can be characterized following bacterial expression and reconstitution of purified apoproteins (Cammarata et al 1990, Paulsen et al 1990, Cammarata and Schmidt 1992, Paulsen and Hobe 1992. From analyses of the products of cab gene deletion mutants, it was shown that amino acids outside the exposed a-helical regions at the NH 2-and COOH-termini of LHCII are not important for the binding and normal spectroscopic properties of the pigments associated with authentic CP2 complexes Schmidt 1992, Paulsen andHobe 1992).…”

Section: Introductionmentioning

confidence: 99%

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Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

1992

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The structure and heterogeneity of LHC II were studied by in vitro reconstitution of apoproteins with pigments (Plumley and Schmidt 1987, Proc Natl Acad Sci 84: 146-150). Reconstituted CP 2 complexes purified by LDS-PAGE were subsequently characterized and shown to have spectroscopic properties and pigment-protein compositions and stoichiometries similar to those of authentic complexes. Heterologous reconstitutions utilizing pigments and light-harvesting proteins from spinach, pea and Chlamydomonas reinhardtii reveal no evidence of specialized binding sites for the unique C. reinhardtii xanthophyll loroxanthin: lutein and loroxanthin are interchangeable for in vitro reconstitution. Proteins modified by the presence of a transit peptide, phosphorylation, or proteolytic removal of the NH2-terminus could be reconstituted. Evidence suggests that post-translational modification are not responsible for the presence of six electrophoretic variants of C. reinhardtii CP 2. Reconstitution is blocked by iodoacetamide pre-treatment of the apoproteins suggesting a role for cysteine in pigment ligation and/or proper folding of the pigment-protein complex. Finally, no effect of divalent cations on pigment reassembly could be detected.

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“…3. A single L H C I I gene product has binding sites for Chls a and b and xanthophylls (Cammarata et al 1990, Paulsen et al 1990, Cammarata and Schmidt 1992. 4.…”

Section: Characterization Of the Pigments And Proteins Which Reconstimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

1992

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“…The minor LHC apoproteins probably first associate with the photosystem II core complexes (CP43, CP47, D1͞2), then monomers of LHCIIb (Lhcb1͞2) form trimers (13) and associate with the growing complex (14). Both chlorophylls and carotenoids are required for proper folding, assembly, and stability of LHC apoproteins (15,16), as demonstrated by detailed in vitro reconstitution studies of pigment-protein complexes (16)(17)(18)(19). Maximum reconstitution efficiency and stability of LHCIIb complexes was achieved when chlorophyll (chl) a, chl b, and the three normal leaf xanthophylls (lutein, violaxanthin, and neoxanthin) were used.…”

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confidence: 99%

“…The apparent localization of lutein in crystallized LHCIIb (32) and its requirement for optimal in vitro assembly of LHCs (16)(17)(18)(19) had led to the assumption that lutein is critical for higher plant photosystem assembly and function. Somewhat surprisingly, Arabidopsis lutein-deficient lut mutants were viable (12,30).…”

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confidence: 99%

Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants

Pogson

Niyogi

Björkman

et al. 1998

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

296

257

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Collectively, the xanthophyll class of carotenoids perform a variety of critical roles in light harvesting antenna assembly and function. The xanthophyll composition of higher plant photosystems (lutein, violaxanthin, and neoxanthin) is remarkably conserved, suggesting important functional roles for each. We have taken a molecular genetic approach in Arabidopsis toward defining the respective roles of individual xanthophylls in vivo by using a series of mutant lines that selectively eliminate and substitute a range of xanthophylls. The mutations, lut1 and lut2 (lut ‫؍‬ lutein deficient), disrupt lutein biosynthesis. In lut2, lutein is replaced mainly by a stoichiometric increase in violaxanthin and antheraxanthin. A third mutant, aba1, accumulates normal levels of lutein and substitutes zeaxanthin for violaxanthin and neoxanthin. The lut2aba1 double mutant completely lacks lutein, violaxanthin, and neoxanthin and instead accumulates zeaxanthin. All mutants were viable in soil and had chlorophyll a͞b ratios ranging from 2.9 to 3.5 and near wild-type rates of photosynthesis. However, mutants accumulating zeaxanthin exhibited a delayed greening virescent phenotype, which was most severe and often lethal when zeaxanthin was the only xanthophyll present. Chlorophyll f luorescence quenching kinetics indicated that both zeaxanthin and lutein contribute to nonphotochemical quenching; specifically, lutein contributes, directly or indirectly, to the rapid rise of nonphotochemical quenching. The results suggest that the normal complement of xanthophylls, while not essential, is required for optimal assembly and function of the light harvesting antenna in higher plants.

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“…We and others have previously attempted to identify pigment binding sites in LHCP by introducing structural alterations such as deletions into the protein and subsequently studying the pigment-binding properties of the protein in v i m . These studies are based on the observation that LHCP and pigments can be reconstituted in vitro to form stable complexes that structurally closely resemble LHCII monomers isolated from thylakoid membranes (Plumley and Schmidt, 1987;Paulsen et al, 1990;Cammarata et al, 1990). We were unable to identify specific pigment binding sites since structural alterations of the prolein would either have virtually no effect on the number or stoichiometry 810 of bound pigments or abolish the formation of stable complexes altogether (Paulsen and Hobe, 1992;Paulsen and Kuttkat, 1993).…”

mentioning

confidence: 99%

Pigments induce folding of light‐harvesting chlorophyll a/b‐binding protein

Paulsen

Finkenzeller

Kühlein

1993

European Journal of Biochemistry

186

131

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The conformational behaviour of the light-harvesting chlorophyll &-binding protein (LHCP), the apoprotein of the major light-harvesting complex (LHCII) of photosystem I1 in plants, has been studied. According to the circular dichroism in the ultraviolet range measured with isolated LHCII, the protein in the complex adopts a folded structure with a high content of a helix (about 60%), whereas the non-pigmented, solubilized protein has a less ordered structure (about 20% a helix).LHCP-pigment complexes that have been reconstituted from the overexpressed protein and isolated pigments in the presence of detergents display a protein CD signal similar to that of authentic LHCII, indicating that LHCP folds into the native structure during the reconstitution procedure. Renaturation of LHCP in these experiments is dependent on the presence of pigments and the formation of stable LHCP-pigment complexes. Pigment-induced engagement of LHCP in a compact structure has also been shown by two additional experimental approaches. (a) Upon complex formation, LHCP or its precursor (pLHCP) becomes resistant to trypsin digestion with the exception of an N-terminal segment of the protein; the same protection of LHCP is known to occur in intact thylakoids. (b) Pigment binding renders a cysteine residue within the N-proximal hydrophobic domain of the protein as well as a newly introduced cysteine four amino acid positions from the C terminus inaccessible to modification with a sulfhydryl-specific label whereas the N terminus stays susceptible to specific labelling. These observations support the notion that only the N terminus protrudes from a compact protein-pigment structure in LHCII. The fact that the major part of LHCP is trypsin-resistant in pigmented complexes reconstituted in the absence of a membrane or even lipids justifies caution in using protection against trypsin as a criterion for the integration of LHCP into the thylakoid membrane.The efficiency of photosynthesis is greatly enhanced by antenna or light-harvesting complexes which collect light energy and transduce it to the photosynthetic reaction centers. The most prominent antenna in higher plants is the major light-harvesting complex of photosystem I1 (LHCII) comprising about half of the total chlorophyll in the thylakoid membrane. LHCII consists of an apoprotein (LHCP) of 25 -28 kDa and pigments, i.e. 14 or 15 molecules chlorophyll a and b and 2 -4 molecules xanthophyll bound/protein monomer (Bassi et al., 1990; Peter and Thornber, 1991). These pigments are non-covalently bound to the apoprotein. Little is known about the molecular interactions between protein and pigments. The light-harvesting function of LHCII requires that captured light quanta must be rapidly transferred between pigments within an antenna to be efficiently delivered at a reaction center (Sauer, 1986). Very rapid energy transfer between antenna chlorophylls has been measured by Abbreviations. LHCII, major light-harvesting complex of photosystem 11; LHCP, light-harvesting chlorophyll &-binding protei...

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Reconstitution of Light Harvesting Complexes: A Single Apoprotein Binds CHLa, CHLb, and Xanthophylls

Cited by 7 publications

References 10 publications

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants

Pigments induce folding of light‐harvesting chlorophyll a/b‐binding protein

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