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E J Boekema

Publications and source records attributed to E J Boekema.

At least 19 recordsLinked to original sources
PubMed2000

Conformational changes in photosystem II supercomplexes upon removal of extrinsic subunits.

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It consists of a large number of intrinsic membrane proteins involved in light-harvesting and electron-transfer processes and of a number of extrinsic proteins required to stabilize photosynthetic oxygen evolution. We studied the structure of dimeric supercomplexes of photosystem II and its associated light-harvesting antenna by electron microscopy and single-particle image analysis. Comparison of averaged projections from native complexes and complexes without extrinsic polypeptides indicates that the removal of 17 and 23 kDa extrinsic subunits induces a shift of about 1.2 nm in the position of the monomeric peripheral antenna protein CP29 toward the central part of the supercomplex. Removal of the 33 kDa extrinsic protein induces an inward shift of the strongly bound trimeric light-harvesting complex II (S-LHCII) of about 0.9 nm, and in addition destabilizes the monomer-monomer interactions in the central core dimer, leading to structural rearrangements of the core monomers. It is concluded that the extrinsic subunits keep the S-LHCII and CP29 subunits in proper positions at some distance from the central part of the photosystem II core dimer to ensure a directed transfer of excitation energy through the monomeric peripheral antenna proteins CP26 and CP29 and/or to maintain sequestered domains of inorganic cofactors required for oxygen evolution.

Buffers↗
PubMed2000

Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membrane of green plant chloroplasts.

The chloroplast thylakoid membrane of green plants is organized in stacked grana membranes and unstacked stroma membranes. We investigated the structural organization of Photosystem II (PSII) in paired grana membrane fragments by transmission electron microscopy. The membrane fragments were obtained by a short treatment of thylakoid membranes with the mild detergent n-dodecyl-alpha, d-maltoside and are thought to reflect the grana membranes in a native state. The membranes frequently show crystalline macrodomains in which PSII is organized in rows spaced by either 26.3 nm (large-spaced crystals) or 23 nm (small-spaced crystals). The small-spaced crystals are less common but better ordered. Image analysis of the crystals by an aperiodic approach revealed the precise positions of the core parts of PSII in the lattices, as well as features of the peripheral light-harvesting antenna. Together, they indicate that the so-called C(2)S(2) and C(2)S(2)M supercomplexes form the basic motifs of the small-spaced and large-spaced crystals, respectively. An analysis of a pair of membranes with a well-ordered large-spaced crystal reveals that many PSII complexes in one layer face only light-harvesting complexes (LHCII) in the other layer. The implications of this type of organization for the efficient transfer of excitation energy from LHCII to PSII and for the stacking of grana membranes are discussed.

Crystallization↗
PubMed2000

Stator structure and subunit composition of the V(1)/V(0) Na(+)-ATPase of the thermophilic bacterium Caloramator fervidus.

The V-type Na(+)-ATPase of the thermophilic, anaerobic bacterium Caloramator fervidus was purified to homogeneity. The subunit compositions of the catalytic V(1) and membrane-embedded V(0) parts were determined and the structure of the enzyme complex was studied by electron microscopy. The V(1) headpiece consists of seven subunits present in one to three copies, and the V(0) part of two subunits in a ratio of 5:2. An analysis of over 7500 single particle images obtained by electron microscopy of the purified V(1)V(0) enzyme complex revealed that the stalk region, connecting the V(1) and V(0) parts, contains two peripheral stalks in addition to a central stalk. One of the two is connected to the V(0) part, while the other is connected to the first via a bar-like structure that is positioned just above V(0), parallel with the plane of the membrane. In projection, this bar seems to contact the central stalk. The data show that the stator structure that prevents rotation of the static part of V(0) relative to V(1) in the rotary catalysis mechanism of energy coupling in ATPases/ATPsynthases is more complex than previously thought.

Adenosine Triphosphatases↗
PubMed1999

Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes.

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It utilizes light for photochemical energy conversion, and is heavily involved in the regulation of the energy flow. We investigated the structural organization of photosystem II and its associated light-harvesting antenna by electron microscopy, multivariate statistical analysis, and classification procedures on partially solubilized photosystem II membranes from spinach. Observation by electron microscopy shortly after a mild disruption of freshly prepared membranes with the detergent n-dodecyl-alpha,D-maltoside revealed the presence of several large supramolecular complexes. In addition to the previously reported supercomplexes [Boekema, E. J., van Roon, H., and Dekker, J. P. (1998) FEBS Lett. 424, 95-99], we observed complexes with the major trimeric chlorophyll a/b protein (LHCII) in a third, L-type of binding position (C2S2M0-2L1-2), and two different types of megacomplexes, both identified as dimeric associations of supercomplexes with LHCII in two types of binding sites (C4S4M2-4). We conclude that the association of photosystem II and its associated light-harvesting antenna is intrinsically heterogeneous, and that the minor CP26 and CP24 proteins play a crucial role in the supramolecular organization of the complete photosystem. We suggest that different types of organization form the structural basis for photosystem II to specifically react to changing light and stress conditions, by providing different routes of excitation energy transfer.

Chloroplasts↗
PubMed1999

Supramolecular organization of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes.

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) Biochemistry 38, 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 degrees and -50 degrees, 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.

Bacterial Proteins↗
PubMed1999

Localization of cyanobacterial photosystem II donor-side subunits by electron microscopy and the supramolecular organization ofphotosystem II in the thylakoid membrane.

A large set of electron microscopy projections of photosystem II (PSII) dimers isolated from the cyanobacterium Synechococcus elongatus was characterized by single particle image analysis. In addition to previously published maps at lower resolution [Boekema, E.J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A.F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179], the new side-view projections show densities of all three lumenal extrinsic proteins, i.e. the 33-kDa, 12-kDa and the cytochrome c-550 subunit encoded by psbO, psbU and psbV, respectively. Analysis of the size and shape of the top-view projections revealed a small number of photosystem II particles of about double the size of the usual dimers. Size and quantity of these 'double dimers' correlates with a small fraction of 1000-kDa particles found with HPLC-size-exclusion chromatographic analysis. Because many cyanobacteria contain dimeric photosystem II complexes arranged in rows within the membrane, the double dimers can be considered as the breakdown fragments of these rows. Their analysis enabled the detection of the arrangement of photosystem II within the rows, in which the dimers interact with other dimers mostly with their tips, leaving a rather open center at the interfaces of two dimers. The dimers have a repeating distance of only 11.7 nm. As a consequence, the phycobilisomes, located on top of PSII and functioning in light-harvesting, must be closely packed or almost touch each other, in a manner similar to a recently suggested model [Bald, D., Kruip, J. & Rögner, M. (1996) Photosynthesis Res. 49, 103-118].

Chromatography, High Pressure Liquid↗
PubMed1998

Specific association of photosystem II and light-harvesting complex II in partially solubilized photosystem II membranes.

In this study, we report the structural characterization of photosystem II complexes obtained from partially solubilized photosystem II membranes. Direct observation by electron microscopy, within a few minutes after a mild disruption of the membranes with the detergent n-dodecyl-alpha,D-maltoside, revealed the presence of several large supramolecular complexes. Images of these complexes were subjected to multivariate statistical analysis and classification procedures, resolving a new complex consisting of the previously characterized dimeric supercomplex of photosystem II and light-harvesting complex II [Boekema et al., Proc. Natl. Acad. Sci. USA 92 (1995) 175-179] and two additional, symmetrically organized protein masses each containing a second type of trimeric light-harvesting II complex. We conclude that large and labile integral membrane proteins, such as photosystem II, can be quickly structurally characterized without extensive purification.

Membrane Proteins↗
PubMed1998

Localization of the 23-kDa subunit of the oxygen-evolving complex of photosystem II by electron microscopy.

A dimeric photosystem II light-harvesting II super complex (PSII-LHCII SC), isolated by sucrose density gradient centrifugation, was previously structurally characterized [Boekema, E. J., Hankamer, B., Bald, D., Kruip, J., Nield, J., Boonstra, A. F., Barber, J. & Rögner, M. (1995) Proc. Natl Acad. Sci. USA 92, 175-179]. This PSII-LHCII SC bound the 33-kDa subunit of the oxygen-evolving complex (OEC), but lacked the 23-kDa and 17-kDa subunits of the OEC. Here the isolation procedure was modified by adding 1 M glycine betaine (1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt) to the sucrose gradient mixture. This procedure yielded PSII-LHCII SC that contained both the 33-kDa and the 23-kDa subunits and had twice the oxygen-evolving capacity of the super complexes lacking the 23-kDa polypeptide. Addition of CaCl2 to PSII-LHCII SC with the 23-kDa subunit attached did not increase the oxygen-evolution rate. This suggests that the 23-kDa subunit is bound in a functional manner and is present in significant amounts. Over 5000 particle projections extracted from electron microscope images of negatively stained PSII-LHCII SC, isolated in the presence and absence of glycine betaine, were analyzed using single-particle image-averaging techniques. Both the 23-kDa and 33-kDa subunits could be visualized in top-view and side-view projections. In the side view the 23-kDa subunit is seen to protrude 0.5-1 nm further than the 33-kDa subunit, giving the PSII particle a maximal height of 9.5 nm. Measured from the centres of the masses, the two 33-kDa subunits associated with the dimeric PSII-LHCII SC are separated by 6.3 nm. The corresponding distance between the two 23-kDa subunits is 8.8 nm.

Betaine↗
PubMed1998

A 7.4-A projection structure of outer membrane phospholipase A from Escherichia coli by electron crystallography.

Outer membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Escherichia coli. Two-dimensional crystals of OMPLA were grown by reconstitution of purified protein into lipid bilayers via detergent dialysis and were studied by electron crystallography. A 7.4-A projection map reveals OMPLA molecules exhibiting an oval-shaped domain of 30 x 20 A resembling the beta-barrel structure characteristic of porins, which is associated with a 25-A elongated domain of lower density.

Bacterial Outer Membrane Proteins↗
PubMed1997

Visualization of a peripheral stalk in V-type ATPase: evidence for the stator structure essential to rotational catalysis.

F- and V-type ATPases are central enzymes in energy metabolism that couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes. They consist of a soluble headpiece that contains the catalytic sites and an integral membrane-bound part that conducts the ion flow. Energy coupling is thought to occur through the physical rotation of a stalk that connects the two parts of the enzyme complex. This mechanism implies that a stator-like structure prevents the rotation of the headpiece relative to the membrane-bound part. Such a structure has not been observed to date. Here, we report the projected structure of the V-type Na+-ATPase of Clostridium fervidus as determined by electron microscopy. Besides the central stalk, a second stalk of 130 A in length is observed that connects the headpiece and membrane-bound part in the periphery of the complex. This additional stalk is likely to be the stator.

Clostridium↗
PubMed1997

Structural organization of the major subunits in cyanobacterial photosystem 1. Localization of subunits PsaC, -D, -E, -F, and -J.

Based on an improved isolation procedure using perfusion chromatography, trimeric Photosystem 1 (PS1) complexes have been isolated from various deletion mutants of the mesophilic cyanobacterium Synechocystis PCC 6803. These mutants are only deficient in the deleted subunits, which was carefully checked by high resolution gel electrophoresis in combination with immunoblotting. These highly purified and well characterized PS1 particles were then examined by electron microscopy, followed by computer-aided image processing with single particle averaging techniques as described earlier (Kruip, J., Boekema, E. J., Bald, D., Boonstra, A. F., and Rögner, M. (1993) J. Biol. Chem. 268, 23353-23360). This precise methodological approach allowed a confident localization of the PS1 subunits PsaC, -D, -E, -F, and -J; it also shows shape and size of these subunits once integrated in the PS1 complex. Subunits PsaC, -D, and -E form a ridge on the stromal site, with PsaE toward the edge of each monomer within the trimer and PsaD extending toward the trimeric center, leaving PsaC in between. PsaF (near PsaE) and PsaJ are close together on the outer edge of each monomer; their proximity is also supported by chemical cross-linking, using the zero-length cross-linker EDC. This localization of PsaF contradicts the position suggested by the published low resolution x-ray analysis and shows for the first time the existence of at least one transmembrane alpha-helix for PsaF. A topographic three-dimensional map has been drawn from this set of results showing the location of the major PS1 subunits (besides PsaA and PsaB). These data also led to the assignment of electron density in the recent medium resolution x-ray structure for PS1 (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973).

Bacterial Proteins↗
PubMed1996

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.

Cyanobacteria↗
PubMed1996

How does photosystem 2 split water? The structural basis of efficient energy conversion.

Photosystem 2 (PS2) is the part of the photosynthetic apparatus that uses light energy to split water releasing oxygen, protons and electrons. Here, we present a model of the subunit organization of PS2 and the accompanying secondary antenna systems (phycobilisomes in cyanobacteria and the light-harvesting complexes in higher plants) and discuss possible physiological consequences of the proposed dimeric structure of PS2.

Biological Evolution↗
PubMed1995

Electron cryomicroscopy of two-dimensional crystals of the H(+)-ATPase from chloroplasts.

The H(+)-ATPase from spinach chloroplasts was isolated and purified. Two-dimensional crystals were obtained from the protein/lipid/detergent micelles by treatment with phospholipase and simultaneous removal of detergent and fatty acids by Biobeads. The resulting two-dimensionally ordered arrays were investigated by electron cryomicroscopy. The ordered arrays showed top view projections of CF0F1. The images were analysed by correlation averaging. In this view CF0F1 has dimensions of 11.4 x 9 nm. The average view shows a strongly asymmetric molecule, in contrast to the rather hexagonal features of CF1, previously analyzed from two-dimensional arrays. It is concluded that this is due either to an asymmetric structure and positioning of CF0 relative to CF1 or to a rearrangement of CF1 subunits induced by binding of CF0 to CF1.

Chloroplasts↗
PubMed1995

Supramolecular structure of the photosystem II complex from green plants and cyanobacteria.

Photosystem II (PSII) complexes, isolated from spinach and the thermophilic cyanobacterium Synechococcus elongatus, were characterized by electron microscopy and single-particle image-averaging analyses. Oxygen-evolving core complexes from spinach and Synechococcus having molecular masses of about 450 kDa and dimensions of approximately 17.2 x 9.7 nm showed twofold symmetry indicative of a dimeric organization. Confirmation of this came from image analysis of oxygen-evolving monomeric cores of PSII isolated from spinach and Synechococcus having a mass of approximately 240 kDa. Washing with Tris at pH 8.0 and analysis of side-view projections indicated the possible position of the 33-kDa extrinsic manganese-stabilizing protein. A larger complex was isolated that contained the light-harvesting complex II (LHC-II) and other chlorophyll a/b-binding proteins, CP29, CP26, and CP24. This LHC-II-PSII complex had a mass of about 700 kDa, and electron microscopy revealed it also to be a dimer having dimensions of about 26.8 and 12.3 nm. From comparison with the dimeric core complex, it was deduced that the latter is located in the center of the larger particle, with additional peripheral regions accommodating the chlorophyll a/b-binding proteins. It is suggested that two LHC-II trimers are present in each dimeric LHC-II-PSII complex and that each trimer is linked to the reaction center core complex by CP24, CP26, and CP29. The results also suggest that PSII may exist as a dimer in vivo.

Cyanobacteria↗
PubMed1994

Electron microscopic structural analysis of Photosystem I, Photosystem II, and the cytochrome b6/f complex from green plants and cyanobacteria.

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochrome b6/f complex are discussed.

Cyanobacteria↗
PubMed1993

Isolation and structural characterization of monomeric and trimeric photosystem I complexes (P700.FA/FB and P700.FX) from the cyanobacterium Synechocystis PCC 6803.

An isolation procedure was developed for the cyanobacterium Synechocystis 6803 (and 6714) which yields both monomeric and trimeric photosystem I complexes (P700.FA/FB complexes) depleted of the stroma-exposed subunits PsaC, -D, and -E (P700.FX complexes). Analysis by high resolution gel electrophoresis in combination with immunoblotting and N-terminal sequencing reveals the selective and quantitative removal of PsaC, -D, and -E from the P700.FA/FB complex, containing PsaA, -B, -C, -D, -E, -F, -K, -L and at least two subunits < or = 4 kDa. Monomeric and trimeric P700.FX complexes show an identical subunit composition and an identical charge recombination half-time of 750 +/- 250 microseconds as determined by flash-induced absorption change measurements, reflecting the quantitative loss of iron-sulfur clusters FA/FB and the presence of cluster FX. The existence of a stable trimeric P700.FX complex enables a detailed structural analysis by electron microscopy with high resolution. Comparison of averaged top and side view projections of P700.FX and P700.FA/FB complexes show that the height of the complex is reduced by about 2.5-3.3 nm upon removal of the three stroma-exposed subunits and indicate the position of these three subunits on the PS I surface. While the outer contours of the stroma exposed mass of PS I agree very well with the three-dimensional crystal analysis recently published for trimeric PS I of Synechococcus elongatus (Krauss, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W., Dauter, Z., Betzel, C., Wilson, K. S., Witt, H. T., and Saenger, W. (1993) Nature 361, 326-330), only the structural analysis presented here is able to assign the stroma-exposed mass exclusively to the subunits PsaC, -D, and -E and to exclude a contribution of other subunits.

Amino Acid Sequence↗