The light reactions of photosynthesis, which include lightharvesting and charge separation, take place in the amphiphilic environment of the thylakoid membrane. The light-harvesting complex II (LHCII) is the main responsible for light absorption in plants and green algae and is involved in photoprotective mechanisms that regulate the amount of excited states in the membrane. The dual function of LHCII has been extensively studied in detergent micelles, but recent results have indicated that the properties of this complex differ in a lipid environment. In this work we checked these suggestions by studying LHCII in liposomes. By combining bulk and single molecule measurements, we monitored the fluorescence characteristics of liposomes containing single complexes up to densely packed proteoliposomes. We show that the natural lipid environment per se does not alter the properties of LHCII, which for single complexes remain very similar to that in detergent. However, we show that LHCII has the strong tendency to cluster in the membrane and that protein interactions and the extent of crowding modulate the lifetimes of the excited state in the membrane. Finally, the presence of LHCII monomers at low concentrations of complexes per liposome is discussed.Photosynthetic organisms evolved the capacity to harvest the energy of solar radiation and store it into chemical compounds. In vascular plants and green algae, sunlight is absorbed by a series of membrane proteins called light-harvesting complexes (LHC).3 The most abundant of these pigment-protein complexes is LHCII. The LHCs have a dual function; in low light conditions they absorb solar energy and efficiently transfer the excitation energy to the reaction center, and in high light they additionally play a role in photoprotection by dissipating the energy absorbed in excess as heat (1, 2). This last process called non-photochemical quenching (NPQ) leads to a decrease of the excited state lifetime of chlorophyll a (Chl a), limiting the possibility of Chl triplet formation and thus the production of singlet oxygen (3). The fast, on the timescale of seconds, and fully reversible part of NPQ is called qE. This mechanism is triggered by the acidification of the lumenal space of the thylakoids, which activates PsbS (4) and LhcSR (5), the proteins responsible for NPQ in plants and green algae, respectively, and the violaxanthin de-epoxidase, which converts violaxanthin into zeaxanthin (for reviews, see Refs. 6 and 7). Although the precise molecular mechanism of quenching has not been fully elucidated yet, a prominent idea discussed in literature is that NPQ is regulated via conformational changes of LHCII. Those changes could be correlated to, or even caused by, LHCII aggregation (8, 9). It was observed that low pH and zeaxanthin, both occurring in high light, enhance LHCII aggregation (10). Aggregation of LHCII in vitro is accompanied by a decrease of the Chl a fluorescence yield, indicating an increased rate of energy dissipation (9, 11). The generation of new quenching sit...

Nine genes (LHCBM1-9) encode the major light-harvesting system of Chlamydomonas reinhardtii. Transcriptomic and proteomic analyses have shown that those genes are all expressed albeit in different amounts and some of them only in certain conditions. However, little is known about the properties and specific functions of the individual gene products because they have never been isolated. Here we have purified several complexes from native membranes and/or we have reconstituted them in vitro with pigments extracted from C. reinhardtii. It is shown that LHCBM1 and -M2/7 represent more than half of the LHCBM population in the membrane. LHCBM2/7 forms homotrimers while LHCBM1 seems to be present in heterotrimers. Trimers containing only type I LHCBM (M3/4/6/8/9) were also observed. Despite their different roles, all complexes have very similar properties in terms of pigment content, organization, stability, absorption, fluorescence and excited-state lifetimes. Thus the involvement of LHCBM1 in non-photochemical quenching is suggested to be due to specific interactions with other components of the membrane and not to the inherent quenching properties of the complex. Similarly, the overexpression of LHCBM9 during sulfur deprivation can be explained by its low sulfur content as compared with the other LHCBMs. Considering the highly conserved biochemical and spectroscopic properties, the major difference between the complexes may be in their capacity to interact with other components of the thylakoid membrane.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 1987 1 , who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

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Signal processing and machine learning algorithms for data supported over graphs, require the knowledge of the graph topology. Unless this information is given by the physics of the problem (e.g., water supply networks, power grids), the topology has to be learned from data. Topology identification is a challenging task, as the problem is often ill-posed, and becomes even harder when the graph structure is time-varying. In this paper, we address the problem of dynamic topology identification by building on recent results from time-varying optimization, devising a general-purpose online algorithm operating in non-stationary environments. Because of its iteration-constrained nature, the proposed approach exhibits an intrinsic temporal-regularization of the graph topology without explicitly enforcing it. As a case-study, we specialize our method to the Gaussian graphical model (GGM) problem and corroborate its performance.