Molecular crowding and order in photosynthetic membranes

The integrity and maintenance of the photosynthetic apparatus in thylakoid membranes of higher plants requires lateral mobility of their components between stacked grana thylakoids and unstacked stroma lamellae. Computer simulations based on realistic protein densities suggest serious problems for lateral protein and plastoquinone diffusion especially in grana membranes, owing to strong retardation by protein complexes. It has been suggested that three structural features of grana thylakoids ensure efficient lateral transport: the organization of protein complexes into supercomplexes; the arrangement of supercomplexes into structured assemblies, which facilitates diffusion process in crowded membranes; the limitation of the diameter of grana discs to less than approximately 500 nm, which keeps diffusion times short enough to support regulation of light harvesting and repair of photodamaged photosystem II.     

Protein diffusion and macromolecular crowding in thylakoid membranes

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 +/- 0.4 x 10(-11) cm(2) s(-1)), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.     

Differential Mobility of Pigment-Protein Complexes in Granal and Agranal Thylakoid Membranes of C3 and C4 Plants

The photosynthetic performance of plants is crucially dependent on the mobility of the molecular complexes that catalyze the conversion of sunlight to metabolic energy equivalents in the thylakoid membrane network inside chloroplasts. The role of the extensive folding of thylakoid membranes leading to structural differentiation into stacked grana regions and unstacked stroma lamellae for diffusion-based processes of the photosynthetic machinery is poorly understood. This study examines, to our knowledge for the first time, the mobility of photosynthetic pigment-protein complexes in unstacked thylakoid regions in the C₃ plant Arabidopsis (Arabidopsis thaliana) and agranal bundle sheath chloroplasts of the C₄ plants sorghum (Sorghum bicolor) and maize (Zea mays) by the fluorescence recovery after photobleaching technique. In unstacked thylakoid membranes, more than 50% of the protein complexes are mobile, whereas this number drops to about 20% in stacked grana regions. The higher molecular mobility in unstacked thylakoid regions is explained by a lower protein-packing density compared with stacked grana regions. It is postulated that thylakoid membrane stacking to form grana leads to protein crowding that impedes lateral diffusion processes but is required for efficient light harvesting of the modularly organized photosystem II and its light-harvesting antenna system. In contrast, the arrangement of the photosystem I light-harvesting complex I in separate units in unstacked thylakoid membranes does not require dense protein packing, which is advantageous for protein diffusion.In higher plants, the photosynthetic apparatus is compartmentalized in the specialized chloroplast organelle. The molecular machinery for the primary photosynthetic processes, the sunlight-driven generation of metabolic energy equivalents, is harbored in an intricate thylakoid membrane system within the chloroplasts. Recent improvements in electron tomography have led to three-dimensional models of the complex architecture of thylakoid membranes (Mustárdy and Garab, 2003; Nevo et al., 2009; Austin and Staehelin, 2011; Daum et al., 2010; Kouřil et al., 2011). Although important details about the thylakoid structure are still highly controversial, consensus exists about the overall design of this membrane system. The thylakoid membrane consists of two morphologically distinct domains: strictly stacked cylindrical grana regions with a diameter of 300 to 600 nm are interconnected by unstacked stroma lamellae, thus forming a continuous membrane system. The molecular complexes that catalyze energy transformation are distributed heterogeneously between the stacked and unstacked membrane regions. The majority of the PSII complex and light-harvesting complex II (LHCII) are localized in stacked thylakoid regions, whereas PSI and the ATP-synthase complex are lacking from stacked grana (Staehelin and van der Staay, 1996; Albertsson, 2001; Dekker and Boekema, 2005). It is assumed that the fifth photosynthetic protein complex (cytochrome b₆f complex) is homogenously distributed.An essential feature of the thylakoid membrane system is its high flexibility, which is required for adaptability and maintenance of the photosynthetic machinery in plants. Highly responsive to environmental conditions, both the overall thylakoid architecture (e.g. number of grana discs) and the molecular membrane composition can change remarkably to optimize, protect, and maintain the photosynthetic apparatus (Walters, 2005; Anderson et al., 2008; Chuartzman et al., 2008; Dietzel et al., 2008; Betterle et al., 2009; Johnson et al., 2011). The underlying molecular processes require brisk protein traffic between stacked and unstacked thylakoid domains (Kirchhoff, 2008). The role of grana in these transport-based processes is poorly understood.Although photosynthetic energy conversion is possible without grana (Anderson et al., 2008), the fact that stacked thylakoids are ubiquitous in almost all land plants (with the exception of chloroplasts in bundle sheath [BS] cells in some C₄ plants; see below) highlights the evolutionary pressure to preserve this complex structural feature. Recently, the importance of grana formation was highlighted in Arabidopsis (Arabidopsis thaliana) mutants that lack the GRANA-DEFICIENT CHLOROPLAST1 gene; they grow much slower than the wild type and exhibit seed lethargy due to missing grana formation (Cui et al., 2011). The functional advantages of grana formation have been discussed extensively (Trissl and Wilhelm, 1993; Mullineaux, 2005; Anderson et al., 2008). It was hypothesized that grana could (1) increase the thylakoid membrane area, and the pigment concentration, in chloroplasts, (2) avoid energy spillover from PSII to PSI, (3) regulate the balance of energy distribution between PSII and PSI by state transition, and (4) enable transversal exciton energy transfer between adjacent grana discs. Although there are good arguments that these possibilities are important for photosynthetic energy conversion, the basis for the evolutionary development of grana has not been determined (Mullineaux, 2005; Anderson et al., 2008).A less considered aspect of grana formation is that it leads to a concentration of protein complexes (Murphy, 1986; Kirchhoff, 2008). The membrane area fraction that belongs to integral photosynthetic protein complexes is about 70%, making grana discs one of the most crowded biomembranes (Kirchhoff, 2008). Light harvesting by PSII benefits from a high protein-packing density for two reasons. First, a concentration of PSII and LHCII in grana ensures a high concentration of light-absorbing pigments that increase the probability of capturing sunlight, which is a “dilute” energy source on the molecular scale (Blankenship, 2002). Second, it has been demonstrated that a high protein-packing density in grana thylakoids is required for efficient intermolecular exciton energy transfer between LHCII and PSII (Haferkamp et al., 2010). Macromolecular crowding ensures that weakly interacting LHCII and PSII complexes come in close contact, allowing efficient Förster-type energy transfer.Besides these advantages, lateral protein traffic is challenged by macromolecular crowding (Mullineaux, 2005; Kirchhoff, 2008). The molecular mobility of proteins in grana thylakoids is reduced by numerous collisions of the diffusing object in the two-dimensional reaction space of the membrane with obstacles, integral membrane proteins, that increase the apparent diffusion path and, consequently, the diffusion time. The strong impairment of a high protein density in grana thylakoids on protein mobility was demonstrated by computer simulations (Tremmel et al., 2003; Kirchhoff et al., 2004) and by diffusion measurement on isolated grana membranes (Kirchhoff et al., 2008) and intact chloroplasts (Goral et al., 2010) using the fluorescence recovery after photobleaching (FRAP) technique. Processes that are expected to be affected by restricted protein mobility are a regulation of energy distributed between PSII and PSI by state transitions (Lemeille and Rochaix, 2010), the repair of photodamaged PSII (Mulo et al., 2008), membrane remodeling triggered by long-term environmental changes (Walters, 2005; Anderson et al., 2008), and the biogenesis of the thylakoid membrane network (Adam et al., 2011). Recently, evidence has accumulated that photoprotective high-energy quenching also requires large-scale diffusion-based structural reorganization within grana thylakoids (Betterle et al., 2009; Johnson et al., 2011).In contrast to our current understanding of diffusion-based processes in thylakoid membranes, knowledge about the factors that determine the mobility of photosynthetic protein complexes in different thylakoid domains is still fragmentary (Mullineaux, 2008). The protein-packing density is very likely a main element that determines protein mobility (Kirchhoff et al., 2008). However, other factors, like electrostatic interactions between proteins by membrane surface charges (Tremmel et al., 2005) or the size and molecular shape of protein complexes (Tremmel et al., 2003), can contribute significantly. However, data only exist about protein mobility for isolated grana thylakoids (Kirchhoff et al., 2008) and for chloroplasts from the grana-containing C₃ plant Arabidopsis (Goral et al., 2010). The diffusion characteristics of the latter are almost completely determined by granal proteins. Limiting information on protein diffusion exists for stroma lamellae of C₃ plants (Consoli et al., 2005; Vladimirou et al., 2009), and no data are available for agranal thylakoids, which occur in BS cells of some C₄ species.This study fills this gap in the knowledge base by studying lateral protein diffusion in unstacked thylakoid membranes in BS chloroplasts of two NADP-malate enzyme (ME)-type C₄ species, maize (Zea mays) and sorghum (Sorghum bicolor), in comparison with the grana-containing mesophyll (M) chloroplasts. The analysis was also complemented by studies on isolated thylakoid subfragments (grana core, grana, and stroma lamellae) from Arabidopsis. The protein mobility was measured by FRAP (Mullineaux and Kirchhoff, 2007), which has been shown to be a straightforward method to analyze protein diffusion in photosynthetic membranes by utilizing natural chlorophyll fluorescence (Kirchhoff et al., 2008; Goral et al., 2010). The comparison with diffusion characteristics in unstacked versus stacked membrane areas highlights the significance of grana formation on the lateral mobility of photosynthetic pigment-protein complexes.     

Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that protein diffusion in the lumen is very restricted

The thylakoid lumen is an important photosynthetic compartment which is the site of key steps in photosynthetic electron transport. The fluidity of the lumen could be a major constraint on photosynthetic electron transport rates. We used Fluorescence Recovery After Photobleaching in cells of the cryptophyte alga Rhodomonas salina to probe the diffusion of phycoerythrin in the lumen and chlorophyll complexes in the thylakoid membrane. In neither case was there any detectable diffusion over a timescale of several minutes. This indicates very restricted phycoerythrin mobility. This may be a general feature of protein diffusion in the thylakoid lumen.     

Relationship between chloroplast structure and O2 evolution rate of leaf discs in plants from different biotopes in South Finland

Abstract The chloroplast ultrastructure, especially the thylakoid organization, the polypeptide composition of the thylakoid membranes and photosynthetic O₂ evolution rate, chlorophyll (Chl) content and Chi a/b ratio were studied in leaves of nine plants growing in contrasting biotopes in the wild in South Finland. All the measurements were made at the beginning of the period of main growth on leaves approaching full expansion, when the CO₂-saturated O₂ evolution rate (measured at 20°C and 1500 μmol photons m^?2s^?1) was at a maximum, ranging from 19.2 to 6.9 μmol O₂ cm^?2 h^?1. Among the species, the Chi a/b ratio varied between 3.75 and 2.71. In the mesophyll chloroplasts, the ratio of the total length of appressed to non-appressed thylakoid membranes varied between 1.07 and 1.79, the number of partitions per granum varied between 2.8 and 12.0 and the grana area between 21 and 42% of the chloroplast area. There was a significant relationship between the rate of O₂ evolution of the leaf discs and the thylakoid organization in the mesophyll chloroplasts. The higher the O₂ evolution rate, the lower was the ratio of the total length of appressed to non-appressed thylakoid membranes and also the lower the grana area. Although the relationship of the photosynthetic rate with the Chi content and the Chi a/b ratio of the leaves was not as clear, a significant negative correlation existed between the Chi a/b ratio and the ratio of appressed to non-appressed thylakoid membranes, indicating lateral heterogeneity in the distribution of different Chl- protein complexes.     

An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked 'grana' regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.     

Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg²⁺ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.     

The structural and functional domains of plant thylakoid membranes

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b₆f complex, and ATPase while depleted in photosystems and light‐harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.     

Thylakoid membrane remodeling during state transitions in Arabidopsis

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.     

Mobility of photosynthetic proteins

The mobility of photosynthetic proteins represents an important factor that affects light-energy conversion in photosynthesis. The specific feature of photosynthetic proteins mobility can be currently measured in vivo using advanced microscopic methods, such as fluorescence recovery after photobleaching which allows the direct observation of photosynthetic proteins mobility on a single cell level. The heterogeneous organization of thylakoid membrane proteins results in heterogeneity in protein mobility. The thylakoid membrane contains both, protein-crowded compartments with immobile proteins and fluid areas (less crowded by proteins), allowing restricted diffusion of proteins. This heterogeneity represents an optimal balance as protein crowding is necessary for efficient light-energy conversion, and protein mobility plays an important role in the regulation of photosynthesis. The mobility is required for an optimal light-harvesting process (e.g., during state transitions), and also for transport of proteins during their synthesis or repair. Protein crowding is then a key limiting factor of thylakoid membrane protein mobility; the less thylakoid membranes are crowded by proteins, the higher protein mobility is observed. Mobility of photosynthetic proteins outside the thylakoid membrane (lumen and stroma/cytosol) is less understood. Cyanobacterial phycobilisomes attached to the stromal side of the thylakoid can move relatively fast. Therefore, it seems that stroma with their active enzymes of the Calvin–Benson cycle, are a more fluid compartment in comparison to the rather rigid thylakoid lumen. In conclusion, photosynthetic protein diffusion is generally slower in comparison to similarly sized proteins from other eukaryotic membranes or organelles. Mobility of photosynthetic proteins resembles restricted protein diffusion in bacteria, and has been rationalized by high protein crowding similar to that of thylakoids.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Insect-induced cecidomyiid galls deficient in light-harvesting protein complex II showing normal grana stacking

It has been postulated that grana stacking and destacking are mediated by the alteration of the surface charge density on the thylakoid membrane, which is regulated by the LHCII phosphorylation and dephosphorylation cycle. Insect-induced cecidomyiid galls, transformed from Machilus thunbergii Sieb & Zucc. (Lauraceae) leaves, are totally deficient in the pigment–protein complexes CPI and A1 of PSI and AB1 and AB2 of PSII. Although the galls are deficient in LHCII complex, grana stacking is normal. Our data suggest that the LHCII complex may not be the only factor responsible for grana stacking of thylakoid membranes.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes

The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion.     

Heterogeneity of thylakoid membranes studied by EPR spin probe

A lipophilic nitroxyl radical, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-adamantylacetate, has been applied to EPR spin probe study of chloroplasts and subchloroplast fragments of different types. The latter originate from grana and the grana core regions. The binding of the spin probe to the membranes was revealed by specific changes in a shape of the EPR spectra. A share of membrane-bound spin probe was different for chloroplasts and subchloroplast fragments, as well as its rotational correlation time and apparent enthalpy and entropy activation of nitroxide rotational motion. The binding of the spin probe induced a significant decrease in the amount of the oxidized P700 and changes in the kinetics of its light oxidation and dark recovery. This suggests that one of the sites of nitroxyl radical binding is the nearest surrounding of the pigment-protein complexes of Photosystem I (PSI). Distinctions in mobility of spin probe immobilized by chloroplasts and their fragments can be caused by the different environment of the PSI complexes located in various regions of thylakoid membranes. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 690–698.     

Chloroplast ultrastructure and thylakoid polypeptide composition are affected by different salt concentrations in the halophytic plant Arthrocnemum macrostachyum

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosynthetic complexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied. Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found that the PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunits with a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, as well as the organization of thylakoid membranes were affected by NaCl concentration, with many grana containing few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mM NaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.     

Coexistence of Fluid and Crystalline Phases of Proteins in Photosynthetic Membranes

Photosystem II (PSII) and its associated light-harvesting complex II (LHCII) are highly concentrated in the stacked grana regions of photosynthetic thylakoid membranes. PSII-LHCII supercomplexes can be arranged in disordered packings, ordered arrays, or mixtures thereof. The physical driving forces underlying array formation are unknown, complicating attempts to determine a possible functional role for arrays in regulating light harvesting or energy conversion efficiency. Here, we introduce a coarse-grained model of protein interactions in coupled photosynthetic membranes, focusing on just two particle types that feature simple shapes and potential energies motivated by structural studies. Reporting on computer simulations of the model’s equilibrium fluctuations, we demonstrate its success in reproducing diverse structural features observed in experiments, including extended PSII-LHCII arrays. Free energy calculations reveal that the appearance of arrays marks a phase transition from the disordered fluid state to a system-spanning crystal. The predicted region of fluid-crystal coexistence is broad, encompassing much of the physiologically relevant parameter regime; we propose experiments that could test this prediction. Our results suggest that grana membranes lie at or near phase coexistence, conferring significant structural and functional flexibility to this densely packed membrane protein system.     

Diffusion of molecules and macromolecules in thylakoid membranes

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Coexistence of Fluid and Crystalline Phases of Proteins in Photosynthetic Membranes

Photosystem II (PSII) and its associated light-harvesting complex II (LHCII) are highly concentrated in the stacked grana regions of photosynthetic thylakoid membranes. PSII-LHCII supercomplexes can be arranged in disordered packings, ordered arrays, or mixtures thereof. The physical driving forces underlying array formation are unknown, complicating attempts to determine a possible functional role for arrays in regulating light harvesting or energy conversion efficiency. Here, we introduce a coarse-grained model of protein interactions in coupled photosynthetic membranes, focusing on just two particle types that feature simple shapes and potential energies motivated by structural studies. Reporting on computer simulations of the model’s equilibrium fluctuations, we demonstrate its success in reproducing diverse structural features observed in experiments, including extended PSII-LHCII arrays. Free energy calculations reveal that the appearance of arrays marks a phase transition from the disordered fluid state to a system-spanning crystal. The predicted region of fluid-crystal coexistence is broad, encompassing much of the physiologically relevant parameter regime; we propose experiments that could test this prediction. Our results suggest that grana membranes lie at or near phase coexistence, conferring significant structural and functional flexibility to this densely packed membrane protein system.     

Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone

We have determined the stoichiometric composition of membrane components (lipids and proteins) in spinach thylakoids and have derived the molecular area occupied by these components. From this analysis, the lipid phase diffusion space, the fraction of lipids located in the first protein solvation shell (boundary lipids), and the plastoquinone (PQ) concentration are derived. On the basis of these stoichiometric data, we have analyzed the motion of PQ between photosystem (PS) II and cytochrome (cyt.) bf complexes in this highly protein obstructed membrane (protein area about 70%) using percolation theory. This analysis reveals an inefficient diffusion process. We propose that distinct structural features of the thylakoid membrane (grana formation, microdomains) could help to minimize these inefficiencies and ensure a non-rate limiting PQ diffusion process. A large amount of published evidence supports the idea that higher protein associations exist, especially in grana thylakoids. From the quantification of the boundary lipid fraction (about 60%), we conclude that protein complexes involved in these associations should be spaced by lipids. Lipid-spaced protein aggregations in thylakoids are qualitatively different to previously characterized associations (multisubunit complexes, supercomplexes). We derive a hierarchy of protein and lipid interactions in the thylakoid membrane.