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

Paulsen, Harald; Finkenzeller, Bärbel; Kühlein, Nicola

doi:10.1111/j.1432-1033.1993.tb18096.x

Cited by 188 publications

(138 citation statements)

References 32 publications

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“…Pigments may also play a more active role during the insertion process. Pigment binding initiates the refolding of detergent-denatured LHCP in vitro (12). This observation suggests that pigment-triggered renaturation of LHCP may play a similar role when the protein is inserted into the thylakoid, the binding of pigments and the concomitant refolding of LHCP being the driving force for translocating parts of the protein across the membrane (13).…”

mentioning

confidence: 83%

Light-harvesting Chlorophyll a/b-binding Protein Stably Inserts into Etioplast Membranes Supplemented with Zn-pheophytina/b

Kuttkat¹,

Edhofer²,

Eichacker³

et al. 1997

Journal of Biological Chemistry

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Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Znpheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Znpheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll-or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll-or Znpheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

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mentioning

confidence: 83%

Light-harvesting Chlorophyll a/b-binding Protein Stably Inserts into Etioplast Membranes Supplemented with Zn-pheophytina/b

Kuttkat¹,

Edhofer²,

Eichacker³

et al. 1997

Journal of Biological Chemistry

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“…The distance between these two labels (106/160) is probably defined by the formation (or completion) of helix H3. Earlier CD measurements had revealed that ␣-helix formation in LHCII apoprotein (beyond the helical structure preformed in dodecyl sulfate solution) is dependent on pigment binding (27), and that most but not all of it happens during the faster reaction phase in the range of 10 s to 1 min. The helix formation positioning labels 106/160 is not likely to be triggered by Chls binding locally to this helical domain, since these are all Chl b binding sites whose occupation takes place during the slower kinetic phase of several minutes.…”

Section: Lhcii Protein Folds In Two Apparent Phases In the Range Of Lmentioning

confidence: 99%

“…(15). For steady-state DEER the spin-labeled complexes were reconstituted using the detergent-exchange procedure (henceforward called ''standard reconstitution'') as described in (27) except that 10 mM ␤-mercaptoethanol (␤-ME) was used as a reductant. Purification of LHCII and preparation of EPR samples were performed as described in (11).…”

Section: Lhcii Protein Folds In Two Apparent Phases In the Range Of Lmentioning

confidence: 99%

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Dockter

Volkov

Bauer

et al. 2009

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

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The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfatesolubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly.assembly kinetics ͉ DEER ͉ LHCII T he major light-harvesting complex LHCII largely increases the efficiency of the photosynthesis process by collecting light energy and conducting it to a photosynthetic reaction center where light-driven charge separation takes place. The apoprotein of LHCII is one of the most abundant membrane proteins on Earth, but even so, our knowledge is fragmentary of how the LHCII apoprotein folds and assembles with pigments. For studying these questions, LHCII offers several advantages. Its crystal structure is known (1), its apoprotein can be recombinantly expressed in Escherichia coli (2), and the protein spontaneously folds and assembles with pigments in detergent solution (2, 3). This spontaneous self-organization can be triggered by mixing the apoprotein solubilized in the ionic detergent dodecyl sulfate with a non-ionic detergent solution of the pigments. The assembly process can then easily be monitored by time-resolved f luorescence spectroscopy using the Chls as built-in fluorescence labels. Such experiments showed that protein folding is dependent on the binding of pigments, and that LHCII formation in vitro occurred in at least two apparent phases, a faster one in the range of some tens of seconds and a slower one taking several minutes (4, 5). The faster step could be assigned to the binding of mostly Chl a, whereas the slower one represents Chl b binding exclus...

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“…WT and mutant apoproteins were isolated from the SG13009 strain of Escherichia coli transformed with constructs following a protocol described previously (17,(21)(22)(23). Reconstitution and purification of protein-pigment complexes were performed as described (11,12).…”

Section: Dna Constructions and Isolation Of Overexpressed Lhcamentioning

confidence: 99%

The Nature of a Chlorophyll Ligand in Lhca Proteins Determines the Far Red Fluorescence Emission Typical of Photosystem I

Morosinotto

Breton

Bassi

et al. 2003

Journal of Biological Chemistry

180

220

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Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at ϳ700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.Photosystem I is a multisubunit pigment-protein complex of the chloroplast membrane acting as a plastocyanin/ferredoxin oxido-reductase in oxygenic photosynthesis. One important spectroscopic feature of PSI 1 is the presence of Chls absorbing at energy lower than the PSI primary electron donor, P700.Although these spectral forms account for only a small percentage of the total absorption, their effect in the energy transfer and trapping of PSI is very prominent (1), with at least 80% of excitation in the complex transiting through them on their way to P700 (2). It has been widely proposed that these forms represent the low energy contributions of excitonic interactions, which involve two or more Chl molecules (3-5); however, the identity of the chromophores involved and the details of the interaction are still unknown.Although the presence of low energy-absorbing Chls is ubiquitous in the PSI of different organisms, their amounts and energies appear to be highly species-dependent (1). In the PSI of higher plants, the red forms are associated with the outer antenna, LHCI (6, 7). LHCI is composed by four pigmentbinding proteins, namely Lhca1-4 (6, 8). These complexes, localized on one side of the core complex (9, 10), are organized in dimers with 10 Chl molecules per subunit (11).As for their properties, Lhca2 and Lhca4 differ from Lhca1 and Lhca3 in many respects. The first two have higher Chl b content with respect to Lhca1 and Lhca3 an...

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

Cited by 188 publications

References 32 publications

Light-harvesting Chlorophyll a/b-binding Protein Stably Inserts into Etioplast Membranes Supplemented with Zn-pheophytina/b

Light-harvesting Chlorophyll a/b-binding Protein Stably Inserts into Etioplast Membranes Supplemented with Zn-pheophytina/b

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

The Nature of a Chlorophyll Ligand in Lhca Proteins Determines the Far Red Fluorescence Emission Typical of Photosystem I

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