Amyloid-ß oligomers have a profound detergent-like effect on lipid membrane bilayers, imaged by atomic force and electron microscopy.

The ability of amyloid-ß peptide (Aß) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aß is reported to have various impacts on the lipid bilayer, but a clearer picture of Aß influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aß assembly types on lipid bilayers. We show that only oligomeric Aß can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aß oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aß monomers and fibers, although Aß fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aß oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

Molecular Recognition: How Photosynthesis Anchors the Mobile Antenna.

True to its name, light-harvesting complex II (LHC II) harvests light energy for photosystem II (PS II). However, LHC II can stray, harvesting light energy for photosystem I (PS I) instead. Cryo-electron microscopy (cryo-EM) now shows how this mobile antenna becomes so attached to its new partner.

Serum Albumin's Protective Inhibition of Amyloid-ß Fiber Formation Is Suppressed by Cholesterol, Fatty Acids and Warfarin.

Central to Alzheimer's disease (AD) pathology is the assembly of monomeric amyloid-ß peptide (Aß) into oligomers and fibers. The most abundant protein in the blood plasma and cerebrospinal fluid is human serum albumin. Albumin can bind to Aß and is capable of inhibiting the fibrillization of Aß at physiological (µM) concentrations. The ability of albumin to bind Aß has recently been exploited in a phase II clinical trial, which showed a reduction in cognitive decline in AD patients undergoing albumin-plasma exchange. Here we explore the equilibrium between Aß monomer, oligomer and fiber in the presence of albumin. Using transmission electron microscopy and thioflavin-T fluorescent dye, we have shown that albumin traps Aß as oligomers, 9 nm in diameter. We show that albumin-trapped Aß oligomeric assemblies are not capable of forming ion channels, which suggests a mechanism by which albumin is protective in Aß-exposed neuronal cells. In vivo albumin binds a variety of endogenous and therapeutic exogenous hydrophobic molecules, including cholesterol, fatty acids and warfarin. We show that these molecules bind to albumin and suppress its ability to inhibit Aß fiber formation. The interplay between Aß, albumin and endogenous hydrophobic molecules impacts Aß assembly; thus, changes in cholesterol and fatty acid levels in vivo may impact Aß fibrillization, by altering the capacity of albumin to bind Aß. These observations are particularly intriguing given that high cholesterol or fatty acid diets are well-established risk factors for late-onset AD.

Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2.

Two-component signal transduction systems (TCSs) consist of sensor histidine kinases and response regulators. TCSs mediate adaptation to environmental changes in bacteria, plants, fungi and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all known cyanobacteria and as chloroplast sensor kinase in eukaryotic algae and plants. Sodium ions have been shown to inhibit the autophosphorylation activity of Hik2 that precedes phosphoryl transfer to response regulators, but the mechanism of inhibition has not been determined. We report on the mechanism of Hik2 activation and inactivation probed by chemical cross-linking and size exclusion chromatography together with direct visualisation of the kinase using negative-stain transmission electron microscopy of single particles. We show that the functional form of Hik2 is a higher-order oligomer such as a hexamer or octamer. Increased NaCl concentration converts the active hexamer into an inactive tetramer. The action of NaCl appears to be confined to the Hik2 kinase domain.

Redox Tuning in Photosystem II.

In photosynthesis, oxygen is liberated from water, not from CO2; however, this model has been silent on why photosynthesis requires bicarbonate. Rutherford and colleagues solve this problem elegantly: bicarbonate tunes water-oxidising photosystem II to make onward electron transfer efficient; an absence of bicarbonate retunes, redirects, and safely shuts down energy flow.

The N-terminal sequence of the extrinsic PsbP protein modulates the redox potential of Cyt b559 in photosystem II.

The PsbP protein, an extrinsic subunit of photosystem II (PSII) in green plants, is known to induce a conformational change around the catalytic Mn4CaO5 cluster securing the binding of Ca(2+) and Cl(-) in PSII. PsbP has multiple interactions with the membrane subunits of PSII, but how these affect the structure and function of PSII requires clarification. Here, we focus on the interactions between the N-terminal residues of PsbP and the α subunit of Cytochrome (Cyt) b559 (PsbE). A key observation was that a peptide fragment formed of the first N-terminal 15 residues of PsbP, 'pN15', was able to convert Cyt b559 into its HP form. Interestingly, addition of pN15 to NaCl-washed PSII membranes decreased PSII's oxygen-evolving activity, even in the presence of saturating Ca(2+) and Cl(-) ions. In fact, pN15 reversibly inhibited the S1 to S2 transition of the OEC in PSII. These data suggest that pN15 can modulate the redox property of Cyt b559 involved in the side-electron pathway in PSII. This potential change of Cyt b559, in the absence of the C-terminal domain of PsbP, however, would interfere with any electron donation from the Mn4CaO5 cluster, leading to the possibility that multiple interactions of PsbP, binding to PSII, have distinct roles in regulating electron transfer within PSII.

Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy.

In higher plants, photosystem II (PSII) is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts, where it is present mostly in dimeric form within the grana. Its light-harvesting antenna system, LHCII, is composed of trimeric and monomeric complexes, which can associate in variable number with the dimeric PSII core complex in order to form different types of PSII-LHCII supercomplexes. Moreover, PSII-LHCII supercomplexes can laterally associate within the thylakoid membrane plane, thus forming higher molecular mass complexes, termed PSII-LHCII megacomplexes (Boekema et al. 1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452). In this study, pure PSII-LHCII megacomplexes were directly isolated from stacked pea thylakoid membranes by a rapid single-step solubilization, using the detergent n-dodecyl-α-D-maltoside, followed by sucrose gradient ultracentrifugation. The megacomplexes were subjected to biochemical and structural analyses. Transmission electron microscopy on negatively stained samples, followed by single-particle analyses, revealed a novel form of PSII-LHCII megacomplexes, as compared to previous studies (Boekema et al.1999a, in Biochemistry 38:2233-2239; Boekema et al. 1999b, in Eur J Biochem 266:444-452), consisting of two PSII-LHCII supercomplexes sitting side-by-side in the membrane plane, sandwiched together with a second copy. This second copy of the megacomplex is most likely derived from the opposite membrane of a granal stack. Two predominant forms of intact sandwiched megacomplexes were observed and termed, according to (Dekker and Boekema 2005 Biochim Biophys Acta 1706:12-39), as (C2S2)4 and (C2S2 + C2S2M2)2 megacomplexes. By applying a gel-based proteomic approach, the protein composition of the isolated megacomplexes was fully characterized. In summary, the new structural forms of isolated megacomplexes and the related modeling performed provide novel insights into how PSII-LHCII supercomplexes may bind to each other, not only in the membrane plane, but also between granal stacks within the chloroplast.

Cross-linking evidence for multiple interactions of the PsbP and PsbQ proteins in a higher plant photosystem II supercomplex.

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.

Proteomic characterization and three-dimensional electron microscopy study of PSII-LHCII supercomplexes from higher plants.

In higher plants a variable number of peripheral LHCII trimers can strongly (S), moderately (M) or loosely (L) associate with the dimeric PSII core (C2) complex via monomeric Lhcb proteins to form PSII-LHCII supercomplexes with different structural organizations. By solubilizing isolated stacked pea thylakoid membranes either with the α or ß isomeric forms of the detergent n-dodecyl-D-maltoside, followed by sucrose density ultracentrifugation, we previously showed that PSII-LHCII supercomplexes of types C2S2M2 and C2S2, respectively, can be isolated [S. Barera et al., Phil. Trans. R Soc. B 67 (2012) 3389-3399]. Here we analysed their protein composition by applying extensive bottom-up and top-down mass spectrometry on the two forms of the isolated supercomplexes. In this way, we revealed the presence of the antenna proteins Lhcb3 and Lhcb6 and of the extrinsic polypeptides PsbP, PsbQ and PsbR exclusively in the C2S2M2 supercomplex. Other proteins of the PSII core complex, common to the C2S2M2 and C2S2 supercomplexes, including the low molecular mass subunits, were also detected and characterized. To complement the proteomic study with structural information, we performed negative stain transmission electron microscopy and single particle analysis on the PSII-LHCII supercomplexes isolated from pea thylakoid membranes solubilized with n-dodecyl-α-D-maltoside. We observed the C2S2M2 supercomplex in its intact form as the largest PSII complex in our preparations. Its dataset was further analysed in silico, together with that of the second largest identified sub-population, corresponding to its C2S2 subcomplex. In this way, we calculated 3D electron density maps for the C2S2M2 and C2S2 supercomplexes, approaching respectively 30 and 28Å resolution, extended by molecular modelling towards the atomic level. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Biophysical and genetic analysis of iron partitioning and ferritin function in Drosophila melanogaster.

Metals have vital functions as prosthetic groups in enzymes, but in labile form they can propagate oxidative stress. The primary function of ferritin is to store bioavailable iron in the form of ferrihydrite. In animals, ferritin is also used to traffic and recycle iron, and to modulate intestinal iron absorption. However, the effect of ferritin accumulation on cellular iron bioavailability remains poorly understood. Moreover, putative in vivo interactions of ferritin with other metal ions have been proposed, but their physiological relevance remains unclear. Here, heterozygous mutant and overexpression ferritin strains of Drosophila melanogaster were subjected to dietary iron manipulations to study the dynamics of iron partition between ferritin and other proteins. Quantitative magnetic analysis of whole fly samples indicated that iron loading of the ferritin core varied in the different genotypes. Total paramagnetic iron content, a likely correlate of bioavailable iron, was reduced in flies overexpressing ferritin when compared with control white flies. Further, three-dimensional maps of the ferritin protein shell and iron core were obtained from single particle transmission electron microscopy imaging and confirmed the similarity between Drosophila and Trichoplusia ferritin structures. Purified Drosophila ferritin also contained small amounts of zinc and manganese. Flies that overexpressed ferritin accumulated in their bodies half the amount of manganese compared to their respective controls. Our results indicate that ferritin may be involved in the homeostasis of other divalent metals, besides iron, and that overexpression of ferritin, sometimes employed to rescue neurodegenerative models of disease, serves to limit divalent metal bio-availability in cells.

Subunit organization of a synechocystis hetero-oligomeric thylakoid FtsH complex involved in photosystem II repair.

FtsH metalloproteases are key components of the photosystem II (PSII) repair cycle, which operates to maintain photosynthetic activity in the light. Despite their physiological importance, the structure and subunit composition of thylakoid FtsH complexes remain uncertain. Mutagenesis has previously revealed that the four FtsH homologs encoded by the cyanobacterium Synechocystis sp PCC 6803 are functionally different: FtsH1 and FtsH3 are required for cell viability, whereas FtsH2 and FtsH4 are dispensable. To gain insights into FtsH2, which is involved in selective D1 protein degradation during PSII repair, we used a strain of Synechocystis 6803 expressing a glutathione S-transferase (GST)-tagged derivative (FtsH2-GST) to isolate FtsH2-containing complexes. Biochemical analysis revealed that FtsH2-GST forms a hetero-oligomeric complex with FtsH3. FtsH2 also interacts with FtsH3 in the wild-type strain, and a mutant depleted in FtsH3, like ftsH2(-) mutants, displays impaired D1 degradation. FtsH3 also forms a separate heterocomplex with FtsH1, thus explaining why FtsH3 is more important than FtsH2 for cell viability. We investigated the structure of the isolated FtsH2-GST/FtsH3 complex using transmission electron microscopy and single-particle analysis. The three-dimensional structural model obtained at a resolution of 26 Å revealed that the complex is hexameric and consists of alternating FtsH2/FtsH3 subunits.

A structural phylogenetic map for chloroplast photosynthesis.

Chloroplasts are cytoplasmic organelles and the sites of photosynthesis in eukaryotic cells. Advances in structural biology and comparative genomics allow us to identify individual components of the photosynthetic apparatus precisely with respect to the subcellular location of their genes. Here we present outline maps of four energy-transducing thylakoid membranes. The maps for land plants and red and green algae distinguish protein subunits encoded in the nucleus from those encoded in the chloroplast. We find no defining structural feature that is common to all chloroplast gene products. Instead, conserved patterns of gene location are consistent with photosynthetic redox chemistry exerting gene regulatory control over its own rate-limiting steps. Chloroplast DNA carries genes whose expression is placed under this control.

The composition and structure of photosystem I-associated antenna from Cyanidioschyzon merolae.

Red algae contain two types of light-harvesting antenna systems, the phycobilisomes and chlorophyll a binding polypeptides (termed Lhcr), which expand the light-harvesting capacity of the photosynthetic reaction centers. In this study, photosystem I (PSI) and its associated light-harvesting proteins were isolated from the red alga Cyanidioschyzon merolae. The structural and functional properties of the largest PSI particles observed were investigated by biochemical characterization, mass spectrometry, fluorescence emission and excitation spectroscopy, and transmission electron microscopy. Our data provide strong evidence for a stable PSI complex in red algae that possesses two distinct types of functional peripheral light-harvesting antenna complex, comprising both Lhcr and a PSI-linked phycobilisome sub-complex. We conclude that the PSI antennae system of red algae represents an evolutionary intermediate between the prokaryotic cyanobacteria and other eukaryotes, such as green algae and vascular plants.

Structural and mutational analysis of band 7 proteins in the cyanobacterium Synechocystis sp. strain PCC 6803.

Band 7 proteins, which encompass members of the stomatin, prohibitin, flotillin, and HflK/C protein families, are integral membrane proteins that play important physiological roles in eukaryotes but are poorly characterized in bacteria. We have studied the band 7 proteins encoded by the cyanobacterium Synechocystis sp. strain PCC 6803, with emphasis on their structure and proposed role in the assembly and maintenance of the photosynthetic apparatus. Mutagenesis revealed that none of the five band 7 proteins (Slr1106, Slr1128, Slr1768, Sll0815, and Sll1021) was essential for growth under a range of conditions (including high light, salt, oxidative, and temperature stresses), although motility was compromised in an Slr1768 inactivation mutant. Accumulation of the major photosynthetic complexes in the thylakoid membrane and repair of the photosystem II complex following light damage were similar in the wild type and a quadruple mutant. Cellular fractionation experiments indicated that three of the band 7 proteins (Slr1106, Slr1768, and Slr1128) were associated with the cytoplasmic membrane, whereas Slr1106, a prohibitin homologue, was also found in the thylakoid membrane fraction. Blue native gel electrophoresis indicated that these three proteins, plus Sll0815, formed large (>669-kDa) independent complexes. Slr1128, a stomatin homologue, has a ring-like structure with an approximate diameter of 16 nm when visualized by negative stain electron microscopy. No evidence for band 7/FtsH supercomplexes was found. Overall, our results indicate that the band 7 proteins form large homo-oligomeric complexes but do not play a crucial role in the biogenesis of the photosynthetic apparatus in Synechocystis sp. strain PCC 6803.

Biochemical and structural studies of the large Ycf4-photosystem I assembly complex of the green alga Chlamydomonas reinhardtii.

Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of >1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography-tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wild-type cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 x 185 A; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.

The multidrug resistance efflux complex, EmrAB from Escherichia coli forms a dimer in vitro.

Tripartite efflux systems are responsible for the export of toxins across both the inner and outer membranes of gram negative bacteria. Previous work has indicated that EmrAB-TolC from Escherichia coli is such a tripartite system, comprised of EmrB an MFS transporter, EmrA, a membrane fusion protein and TolC, an outer membrane channel. The whole complex is predicted to form a continuous channel allowing direct export from the cytoplasm to the exterior of the cell. Little is known, however, about the interactions between the individual components of this system. Reconstitution of EmrA+EmrB resulted in co-elution of the two proteins from a gel filtration column indicating formation of the EmrAB complex. Electron microscopic single particle analysis of the reconstituted EmrAB complex revealed the presence of particles approximately 240x140A, likely to correspond to two EmrAB dimers in a back-to-back arrangement, suggesting the dimeric EmrAB form is the physiological state contrasting with the trimeric arrangement of the AcrAB-TolC system.

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize.

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.

Probing the organization of photosystem II in photosynthetic membranes by atomic force microscopy.

Efficient photosynthetic energy transduction and its regulation depend on a precise supramolecular arrangement of the plant photosystem II (PSII) complex in grana membranes of chloroplasts. The topography of isolated photosystem II supercomplexes and the supramolecular organization of this complex in grana membrane preparations are visualized by high-resolution atomic force microscopy (AFM) in air in tapping mode with an active feedback control to minimize tip-sample interactions. Systematic comparison between topographic characteristics of the protrusions in atomic force microscopic images and well-established high-resolution and freeze-fracture electron microscopic data shows that the photosystem II organization can be properly imaged by AFM in air. Taking the protruding water-splitting apparatus as a topographic marker for PSII, its distribution and orientation in isolated grana membrane were analyzed. For the latter a new mathematical procedure was established, which revealed a preference for a parallel alignment of PSII that resembles the organization in highly ordered semicrystalline arrays. Furthermore, by analyzing the height of grana membrane stacks, we conclude that lumenal protrusions of adjacent photosystem II complexes in opposing membranes are displaced relative to each other. The functional consequences for lateral migration processes are discussed.

A highly active histidine-tagged Chlamydomonas reinhardtii Photosystem II preparation for structural and biophysical analysis.

We describe a genetically engineered strain of Chlamydomonas reinhardtii where the PsbH subunit of Photosystem II (PSII) has been modified to include a C-terminal polyhistidine tag. The strain was generated by the rescue to photoautotrophic growth of a psbH insertional mutant following chloroplast transformation with the modified gene. This selection strategy confirms that the addition of the tag to PsbH does not prevent the assembly of functional PSII, and results in an engineered strain with tagged PSII but no antibiotic-resistance markers in the chloroplast genome. Consequently, the strain is suitable for subsequent genetic manipulation of chloroplast PSII genes. We also describe a rapid PSII isolation procedure that gives a preparation capable of high rates of oxygen evolution. This preparation is suitable for spectroscopic analysis as shown by EPR analysis of the S2 state of the water oxidation cycle. Furthermore, electron microscopy, coupled to single particle analysis, has revealed the isolated PSII to be structurally homogeneous core dimers that are ideally suited for higher resolution structural studies.