We present an extended analysis of the organization of green plant photosystem II and its associated lightharvesting 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 2208 and 2508, 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 C 2 S 2 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.Keywords: photosystem II; light harvesting complex; thylakoid membrane; electron microscopy.A key role in the energy-conserving mechanisms of green plants is played by photosystem II (PSII). Its major task is to use light energy for the reduction of plastoquinone, the oxidation of water and the formation of a transmembrane pH gradient. It consists of at least 25 different types of protein subunits [1], which are organized into two structurally and functionally distinct parts. The first part is the core complex, a well-defined structure that is responsible for all electron transfer reactions in PSII, including the formation of oxygen. It contains the reaction centre proteins D1 and D2, cytochrome b-559, two core antenna proteins called CP47 and CP43, a number of extrinsic proteins indirectly involved in the oxygenevolving process, and several small proteins of unknown function [1]. There is overwhelming evidence that the PSII core complex is organized as a dimer in the stacked, appressed regions of the thylakoid membrane [2±4]. The structure of the PSII core complex without the CP43 subunit has been determined at 8 A Ê resolution by electron crystallography on two-dimensional crystals [5,6]. The structure reveals 23 transmembrane a-helices [7], of which six have been assigned to CP47, and 10 to the D1 and D2 proteins. Two of the remaining seven transmembrane a-helices are probably formed by cytochrome b-559, while the others are thought to belong to small proteins consisting of a single transmembrane a-helix [6].The second part of PSII is the peripheral antenna, which in g...

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse‐phase evaporation of octylglucoside‐solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non‐physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n‐dodecyl)ether (C12E8), and subsequent detergent removal with Bio‐Beads SM‐2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH‐dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.

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Isothermal titration microcalorimetry has been applied to investigate the interactions between hydrophobically-modified water-soluble polymers and surfactants. The following polymers were used in this study:  poly(sodium acrylate-co-n-alkyl methacrylate) (A), where n-alkyl = C9H19, C12H25, and C18H37 (percentage of n-alkyl methacrylate to total monomer content ranging from 0 to 8), and poly(acrylamide-co-n-alkyl methacrylate) (B), where n-alkyl = C12H25 (percentage of lauryl methacrylate to total monomer content ranging from 0 to 5). The surfactants were a cyclic (mono-) n-dodecyl sodium phosphate (1) (CMP), a cyclic di-n-dodecyl sodium phosphate (2) (CDP), n-dodecyltrimethylammonium bromide (3) (DTAB), and di-n-dodecyldimethylammonium bromide (4) (DDAB). The following factors were found to influence the interactions between polymers and surfactants:  electrostatic forces, polymer hydrophobicity (both the length of the hydrophobic moiety and the degree of hydrophobic modification), and the aggregational states of the amphiphilic molecules, which are micellar for the single-tailed surfactants and vesicular for the double-tailed amphiphiles. We provide evidence that, in the case of the single-tailed surfactants, individual amphiphilic molecules adsorb onto existing polymeric microdomains. This is in strong contrast with ‘classical' polymer−surfactant interactions, where cooperative aggregation of single-tailed amphiphiles in the presence of homopolymers like poly(ethylene oxide) or poly(propylene oxide) was found at concentrations lower than the critical micelle concentration in pure water. In the case of vesicle-forming surfactants, the hydrophobic side chain of the polymer anchors into the bilayers of the vesicles. Non-hydrophobically-modified polymers do not interact at all with the vesicle bilayers. Interestingly, the interactions between single-tailed surfactants and hydrophobically-modified polymers are governed by different factors than the binding of hydrophobically-modified polymers to vesicular bilayers. In the former case, the number and strength of existing (inter)polymeric associations is of importance, and it is particularly the length of the hydrophobic moieties that is decisive. However, for favorable polymer−bilayer interactions it is sufficient that the hydrophobic moieties are long enough to be able to anchor. If this is the case, the number of hydrophobic anchors per polymer molecule further determines the effectiveness of the interaction. Finally, it appears that electrostatic repulsions can be easily overcome by hydrophobic interactions, but added salt facilitates the interactions between equally charged polymers and surfactants.