In vitro reconstitution of a light-harvesting gene product: deletion mutagenesis and analyses of pigment binding

Cammarata, Kirk V.; Schmidt, Gregory W.

doi:10.1021/bi00125a019

Cited by 62 publications

(58 citation statements)

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“…This is in accordance with earlier observations that carotenoids are required for the formation of LHCIIb in vitro (8,9) with lutein being the most efficiently stabilizing carotenoid (22)(23)(24). Unexpectedly, the fluorescent dye monitor detected specific Chl a binding to Lhcb1 in the absence of Chl b, although it had been noted earlier that Chl b is an absolute requirement for stable Lhcb1-pigment complex formation in vitro (21,22) and stable insertion of Lhcb1 in etioplast membranes (25). It is well established that LHCIIb appearance in greening plant tissue coincides with the accumulation of Chl b (26 -28).…”

Section: Discussionsupporting

confidence: 93%

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Horn

Paulsen

2004

Journal of Biological Chemistry

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The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.The major light-harvesting chlorophyll (Chl) 1 a/b complex (LHCIIb) greatly enhances the efficiency of photosynthesis by enlarging the absorption cross-section of photosystem (PS) II and, under some conditions, also of PS I. The structure of LHCIIb has been known in near-atomic detail since 1994 (1) and recently has been further resolved to 2.7 Å (2). Much less detail is known about some steps in the biogenesis of LHCIIb.The apoprotein, Lhcb1-3, is synthesized in its precursor form in the cytoplasm, imported into the plastid, and finally inserted into the thylakoid membrane via the signal recognition particle pathway (3, 4). It is unclear when and even where the protein becomes complexed with pigments. The signal recognition particle complex presumably keeps Lhcb1-3 in an unfolded and thus nonpigmented form until it is delivered to the thylakoid; on the other hand, Hoober and Eggink (5) proposed, based on observations in the green alga Chlamydomonas reinhardtii, that LHCIIb assembly takes place in the envelope membrane. We do not know how the Chls are delivered to their apoproteins, whether the last steps in their biogenetic pathway take place in the immediate neighborhood of the site of assembly or whether the pigments are transported there via some carrier (6, 7). All we can safely assume is that Chl molecules do not freely diffuse in the thylakoid membrane because this...

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Section: Discussionsupporting

confidence: 93%

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Horn

Paulsen

2004

Journal of Biological Chemistry

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“…The functional reason for the absence of neoxanthin in CP24 and in LHCI proteins is presently unknown. CP24 can only be reconstituted in the presence of carotenoids consistently with previous reports with LHCII (18,23,32) and CP29 (33). However, there is only limited specificity in carotenoid binding.…”

Section: Discussionsupporting

confidence: 87%

“…17 showed that the expression products are accumulated in inclusion bodies, as shown for LHCII (18,32) and CP29 (33). Repeated washings of this pelletable fraction yielded 80% pure CP24 apoprotein as judged by polyacrylamide electrophoresis.…”

Section: Expression Of Maize Cp24 Gene Construct In Bacteria Andmentioning

confidence: 68%

“…Reconstitution of pigment-proteins from recombinant apoprotein and isolated pigments was previously performed in the case of the major LHCII protein (32,42), of two LHCI subunits (49), and of the minor PSII subunit CP29 (33). In the latter case, it was possible to reproduce all of the biochemical and spectroscopic features of the native complex, thus allowing mutation analysis of chlorophyll-binding sites (12,35).…”

Section: Discussionmentioning

confidence: 99%

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In VitroReconstitution of the Recombinant Photosystem II Light-harvesting Complex CP24 and Its Spectroscopic Characterization

Pagano

Cinque

Bassi

1998

Journal of Biological Chemistry

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The light-harvesting chlorophyll a/b protein CP24, a minor subunit of the photosystem II antenna system, is a major violaxanthin-binding protein involved in the regulation of excited state concentration of chlorophyll a. This subunit is poorly characterized due to the difficulty in isolation and instability during purification procedures. We have used an alternative approach in order to gain information on the properties of this protein; the Lhcb6 cDNA has been overexpressed in bacteria in order to obtain the CP24 apoprotein, which was then reconstituted in vitro with xanthophylls, chlorophyll a, and chlorophyll b, yielding a pigment-protein complex with properties essentially identical to the native protein extracted from maize thylakoids. Although all carotenoids were supplied during refolding, the recombinant holoprotein exhibited high selectivity in xanthophyll binding by coordinating violaxanthin and lutein but not neoxanthin or ␤-carotene. Each monomer bound a total of 10 chlorophyll a plus chlorophyll b and two xanthophyll molecules. Moreover, the protein could be refolded in the presence of different chlorophyll a to chlorophyll b ratios for yielding a family of recombinant proteins with different chlorophyll a/b ratios but still binding the same total number of porphyrins. A peculiar feature of CP24 was its refolding capability in the absence of lutein, contrary to the case of other homologous proteins, thus showing higher plasticity in xanthophyll binding. These characteristics of CP24 are discussed with respect to its role in binding zeaxanthin in high light stress conditions.The spectroscopic analysis of a recombinant CP24 complex binding eight chlorophyll b molecules and a single chlorophyll a molecule by Gaussian deconvolution allowed the identification of four subbands peaking at wavelengths of 638, 645, 653, and 659 nm, which have an increased amplitude with respect to the native complex and therefore identify the chlorophyll b absorption in the antenna protein environment. Gaussian subbands at wavelengths 666, 673, 679, and 686 nm are depleted in the high chlorophyll b complex, thus suggesting they derive from chlorophyll a.In higher plants, chloroplasts, chlorophyll, and carotenoid molecules are noncovalently bound to specific transmembrane proteins to form light-harvesting complexes called LHCI 1 and LHCII. These antenna complexes efficiently capture the light and deliver the excitation energy, respectively, to photosystem I (PSI) and II (PSII) reaction centers, where electron transport occurs, yielding a trans-thylakoid pH gradient, ATP synthesis, and NADP ϩ reduction. The photosystem II light-harvesting complex has been extensively investigated and shown to be composed of four chlorophyll a/b proteins, the major complex (LHCII) binding about 65% of PSII chlorophyll and three minor complexes (called CP24, CP26, and CP29) that together bind about 15% of total PSII chlorophyll (1). These minor chlorophyll proteins appear to be involved in the dissipation of the chlorophyll excitation energy nee...

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“…Lutein and b-carotene were identified by comparison with commercial standards. Neoxanthin and violaxanthin standards were isolated by thin-layer chromatography of carotenoids (Cammarata and Schmidt, 1992).…”

Section: Analysis Of Carotenoid Composition Of Y1-8549 Mutantmentioning

confidence: 99%

The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

et al. 2008

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Carotenoids are essential for photosynthesis and photoprotection; they also serve as precursors to signaling molecules that influence plant development and biotic/abiotic stress responses. With potential to improve plant yield and nutritional quality, carotenoids are targets for metabolic breeding/engineering, particularly in the Poaceae (grass family), which includes the major food crops. Depending on genetic background, maize (Zea mays) endosperm carotenoid content varies, and therefore breeding-enhanced carotenoid levels have been of ongoing interest. The first committed step in the plastid-localized biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). The gene family in maize and other grasses contains three paralogs with specialized roles that are not well understood. Maize endosperm carotenoid accumulation requires PSY1 expression. A maize antibody was used to localize PSY1 to amyloplast envelope membranes and to determine PSY1 accumulation in relation to carotenoid accumulation in developing endosperm. To test when and if PSY transcript levels correlated with carotenoid content, advantage was taken of a maize germplasm diversity collection that exhibits genetic and chemical diversity. Total carotenoid content showed statistically significant correlation with endosperm transcript levels at 20 d after pollination for PSY1 but not PSY2 or PSY3. Timing of PSY1 transcript abundance, previously unknown, provides critical information for choosing breeding alleles or properly controlling introduced transgenes. PSY1 was unexpectedly found to have an additional role in photosynthetic tissue, where it was required for carotenogenesis in the dark and for heat stress tolerance. Leaf carotenogenesis was shown to require phytochrome-dependent and phytochrome-independent photoregulation of PSY2 plus nonphotoregulated PSY1 expression.

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In vitro reconstitution of a light-harvesting gene product: deletion mutagenesis and analyses of pigment binding

Cited by 62 publications

References 50 publications

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

In VitroReconstitution of the Recombinant Photosystem II Light-harvesting Complex CP24 and Its Spectroscopic Characterization

The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

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