Assembly of the Major Light-Harvesting Complex II in Lipid Nanodiscs

Section of Biophysics, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
Biophysical Journal (Impact Factor: 3.97). 11/2011; 101(10):2507-15. DOI: 10.1016/j.bpj.2011.09.055
Source: PubMed


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.

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Available from: Willem J Degrip
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    • "Transmission electron microscopy (TEM) and cryo-microscopy (cryo-EM) are very powerful tools to determine the structural organization of proteins and macromolecular complexes6061. TEM, given the size of nanodiscs, is very useful to evaluate samples in terms of size homogeneity and structural integrity[20,62636465. Nanodiscs are usually visualized in TEM images as circular particles with the dimensions expected for the MSP variant, especially in the case of empty nanodiscs or nanodiscs containing a small protein (Fig. 1B)[27,59]. "
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    • "In these measurements, the degree of fluorescence quenching was strongly dependent on the level of aggregation (itself related to the amount of detergent removed from the solution) or on the protein-to-lipid ratio in proteoliposomes . Short components (300–400 ps) were also measured for LHCII incorporated in nanodiscs, but they were proposed to originate from a small number of LHCII aggregates of size \100 nm and to be unrelated to the incorporation of LHCII into nanodiscs (Pandit et al. 2011). Since ultracentrifugation and SEC data exclude the possibility of aggregation of A8-35-trapped LHCII, it can be concluded that the 230-ps component originates from a quenched conformation of trimeric LHCII. "
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    ABSTRACT: The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the "light-harvesting conformation", whereas the "quenched conformation" is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30 % shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.
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    • "Examples of plant membrane proteins reconstituted in nanodiscs are rare. One example of this approach is the isolation of trimeric light-harvesting complex (LHCII) from spinach and its reconstitution into asolectin nanodiscs (Pandit et al. 2011). The absorbance and fluorescence spectra differed between nanodisc and surfactant-solubilized LHCII, implying differences in conformations of LHCII and their micro-environments. "
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