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ABSTRACT: A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid
membranes from spinach with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted
of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its
associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b
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f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and
from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic
structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very
low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms.
Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes.
An extensive analysis by electron microscopy and image analysis of the CF0F1 ATP synthase complex suggests locations of the δ (on top of the F1 headpiece) and ∈ subunits (in the central stalk) and reveals that in a substantial part of the complexes the F1 headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure
and may represent the complex in its inactive, oxidized form.
Photosynthesis Research 04/2012; 64(2):155-166. · 3.24 Impact Factor
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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.
Biophysical Journal 11/2011; 101(10):2507-15. · 3.65 Impact Factor
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ABSTRACT: The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.
Journal of Biological Chemistry 09/2011; 286(45):39164-71. · 4.77 Impact Factor
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ABSTRACT: In many natural habitats, growth of cyanobacteria may be limited by a low concentration of iron. Cyanobacteria respond to this condition by expressing a number of iron-stress-inducible genes, of which the isiA gene encodes a chlorophyll-binding protein known as IsiA or CP43'. IsiA monomers assemble into ring-shaped polymers that encircle trimeric or monomeric photosystem I (PSI), or are present in supercomplexes without PSI, in particular upon prolonged iron starvation. In this report, we present steady-state and time-resolved fluorescence measurements of isolated IsiA aggregates that have been purified from an iron-starved psaFJ-minus mutant of Synechocystis PCC 6803. We show that these aggregates have a fluorescence quantum yield of approximately 2% compared to that of chlorophyll a in acetone, and that the dominating fluorescence lifetimes are 66 and 210 ps, more than 1 order of magnitude shorter than that of free chlorophyll a. Comparison of the temperature dependence of the fluorescence yields and spectra of the isolated aggregates and of the cells from which they were obtained suggests that these aggregates occur naturally in the iron-starved cells. We suggest that IsiA aggregates protect cyanobacterial cells against the deleterious effects of light.
Biochemistry 09/2005; 44(32):10846-53. · 3.42 Impact Factor
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ABSTRACT: We have investigated the structure of the higher plant light harvesting complex of photosystem I (LHCI) by analyzing PSI-LHCI particles isolated from a set of Arabidopsis plant lines, each lacking a specific Lhca (Lhca1-4) polypeptide. Functional antenna size measurements support the recent finding that there are four Lhca proteins per PSI in the crystal structure [Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635]. According to HPLC analyses the number of pigment molecules bound within the LHCI is higher than expected from reconstitution studies or analyses of isolated native LHCI. Comparison of the spectra of the particles from the different lines reveals chlorophyll absorption bands peaking at 696, 688, 665, and 655 nm that are not present in isolated PSI or LHCI. These bands presumably originate from "gap" or "linker" pigments that are cooperatively coordinated by the Lhca and/or PSI proteins, which we have tentatively localized in the PSI-LHCI complex.
Biochemistry 04/2005; 44(8):3065-73. · 3.42 Impact Factor
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ABSTRACT: A highly purified cytochrome b(6)f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b(6)f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b(6), which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b(6)f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9-cis isomers of beta-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid - possibly for strucure and assembly - and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b(6)f complex.
FEBS Journal 02/2005; 272(2):582-92. · 3.79 Impact Factor
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ABSTRACT: We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-alpha,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes.
Photosynthesis Research 02/2002; 72(2):203-10. · 3.24 Impact Factor
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ABSTRACT: We present an extended analysis of the organization of green plant photosystem II and its associated light-harvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII–LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry38, 2233–2239], the following results were obtained.The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about −20° and −50°, respectively.The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions.A new type of megacomplex was observed with a characteristic windmill-like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips.Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins.
European Journal of Biochemistry. 11/1999; 266(2):444 - 452.
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ABSTRACT: The organization of Arabidopsis thaliana photosystem II (PSII) and its associated light-harvesting antenna (LHCII) was studied in isolated PSII–LHCII supercomplexes and native membrane-bound crystals by transmission electron microscopy and image analysis. Over 4000 single-particle projections of PSII–LHCII supercomplexes were analyzed. In comparison to spinach supercomplexes some striking differences were revealed: a much larger number of supercomplexes from Arabidopsis contain copies of M-type LHCII trimers. M-type trimers can also bind in the absence of the more common S-type trimers. No binding of L-type trimers could be detected. Analysis of native membrane-bound PSII crystals revealed a novel type of crystal with a unit cell of 25.6 × 21.4 nm (angle 77°), which is larger than any of the PSII lattices observed before. The data show that the unit cell is built up from C2S2M2 supercomplexes, rather than from C2S2M supercomplexes observed in native membrane crystals from spinach. It is concluded from both the single particle analysis and the crystal analysis that the M-type trimers bind more strongly to PSII core complexes in Arabidopsis than in spinach.
