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Assembly of the major light-harvesting complex II in lipid nanodiscs.

ABSTRACT: Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q(y) region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.

SUBMITTER: Pandit A

PROVIDER: S-EPMC3218333 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Assembly of the major light-harvesting complex II in lipid nanodiscs.

Pandit Anjali A Shirzad-Wasei Nazhat N Wlodarczyk Lucyna M LM van Roon Henny H Boekema Egbert J EJ Dekker Jan P JP de Grip Willem J WJ

Biophysical journal 20111115 10

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we ass ...[more]

PMID: 22098750

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Project description:Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments - protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl a and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chl a photobleaching in membranes and detergent was found to be 3.4 × 10-5 and 1.4 × 10-5, respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 × 10-5 and 2 × 10-5). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibition in vivo. The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage.

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Assembly of the major light-harvesting complex II in lipid nanodiscs.

Publications

Assembly of the major light-harvesting complex II in lipid nanodiscs.

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