The Structural and Spectral Features of Light-Harvesting Complex II Proteoliposomes Mimic Those of Native Thylakoid Membranes.
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ABSTRACT:
SUBMITTER: Wilson S
PROVIDER: S-EPMC9237827 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The light-harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single-particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P-LHCII) conformations. These are part of an important, reversible, energy re-equilibration process called the state transition. We found that the population of P-LHCII in unappressed membranes is more mobile than the population of non-P-LHCII from the same regions.
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Project description:The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, "transit complex", and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.
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Project description:Interaction forces of membrane protein subunits are of importance in their structure, assembly, membrane insertion, and function. In biological membranes, and in the photosynthetic apparatus as a paradigm, membrane proteins fulfill their function by ensemble actions integrating a tight assembly of several proteins. In the bacterial photosynthetic apparatus light-harvesting complexes 2 (LH2) transfer light energy to neighboring tightly associated core complexes, constituted of light-harvesting complexes 1 (LH1) and reaction centers (RC). While the architecture of the photosynthetic unit has been described, the forces and energies assuring the structural and functional integrity of LH2, the assembly of LH2 complexes, and how LH2 interact with the other proteins in the supramolecular architecture are still unknown. Here we investigate the molecular forces of the bacterial LH2 within the native photosynthetic membrane using atomic force microscopy single-molecule imaging and force measurement in combination. The binding between LH2 subunits is fairly weak, of the order of k(B)T, indicating the importance of LH2 ring architecture. In contrast LH2 subunits are solid with a free energy difference of 90 k(B)T between folded and unfolded states. Subunit ?-helices unfold either in one-step, ?- and ?-polypeptides unfold together, or sequentially. The unfolding force of transmembrane helices is approximately 150 pN. In the two-step unfolding process, the ?-polypeptide is stabilized by the molecular environment in the membrane. Hence, intermolecular forces influence the structural and functional integrity of LH2.
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Project description:Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these membranes. Combined with a new method to visualize membrane surface topology, we map the molecular landscapes of thylakoid membranes inside green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.
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Project description:The light-harvesting chlorophyll a/b binding proteins (LHCP) belong to a large family of membrane proteins. They form the antenna complexes of photosystem I and II and function in light absorption and transfer of the excitation energy to the photosystems. As nuclear-encoded proteins, the LHCPs are imported into the chloroplast and further targeted to their final destination-the thylakoid membrane. Due to their hydrophobicity, the formation of the so-called 'transit complex' in the stroma is important to prevent their aggregation in this aqueous environment. The posttranslational LHCP targeting mechanism is well regulated through the interaction of various soluble and membrane-associated protein components and includes several steps: the binding of the LHCP to the heterodimeric cpSRP43/cpSRP54 complex to form the soluble transit complex; the docking of the transit complex to the SRP receptor cpFtsY and the Alb3 translocase at the membrane followed by the release and integration of the LHCP into the thylakoid membrane in a GTP-dependent manner. This review summarizes the molecular mechanisms and dynamics behind the posttranslational LHCP targeting to the thylakoid membrane of Arabidopsis thaliana.
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Project description:How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4 +/- 2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the nonaggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4 +/- 2 degrees from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knockout, LH1 knockout, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and nonaggregated LH2 proteins in the photosynthetic membranes.
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Project description:Light-harvesting complex II (LHCII) is a pigment-protein complex present in higher plants and green algae. LHCII represents the main site of light absorption, and its role is to transfer the excitation energy toward the photosynthetic reaction centers, where primary energy conversion reactions take place. The optical properties of LHCII are known to depend on protein conformation. However, the relation between the structural and spectroscopic properties of the pigments is not fully understood yet. In this respect, previous classical molecular dynamics simulations of LHCII in a model membrane [Sci. Rep. 2015, 5, 1-10] have shown that the configuration and excitonic coupling of a chlorophyll (Chl) dimer functioning as the main terminal emitter of the complex are particularly sensitive to conformational changes. Here, we use quantum chemistry calculations to investigate in greater detail the effect of pigment-pigment interactions on the excited-state landscape. While most previous studies have used a local picture in which electrons are localized on single pigments, here we achieve a more accurate description of the Chl dimer by adopting a supramolecular picture where time-dependent density functional theory is applied to the whole system at once. Our results show that specific dimer configurations characterized by shorter inter-pigment distances can result in a sizable intensity decrease (up to 36%) of the Chl absorption bands in the visible spectral region. Such a decrease can be predicted only when accounting for Chl-Chl charge-transfer excitations, which is possible using the above-mentioned supramolecular approach. The charge-transfer character of the excitations is quantified by two types of analyses: one focusing on the composition of the excitations and the other directly on the observable total absorption intensities.
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Project description:The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions.
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Project description:Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.
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Project description:Plant light-harvesting complex II (LHCII) is the key antenna complex for plant photosynthesis. We present coarse-grained molecular dynamics simulations of monomeric and trimeric LHCII in a realistic thylakoid membrane environment based on the Martini force field. The coarse-grained protein model has been optimized with respect to atomistic reference simulations. Our simulations provide detailed insights in the thylakoid lipid fingerprint of LHCII which compares well with experimental data from membrane protein purification. Comparing the monomer and trimeric LHCII reveals a stabilizing effect of trimerization on the chromophores as well as the protein. Moreover, the average chromophore distance shortens in the trimer leading to stronger excitonic couplings. When changing the native thylakoid environment to a model membrane the protein flexibility remains constant, whereas the chromophore flexibility is reduced. Overall, the presented LHCII model lays the foundation to investigate the ?s dynamics of this key antenna protein of plants.
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