Progress towards structural elucidation of Photosystem II

In recent years Photosystem II, and in particular the oxygen evolving component of the enzyme, have been the subject of intense biochemical and biophysical analysis. To date no high resolution structural model of the complex has been produced. As a consequence unambiguous interpretation of much experimental data has proven difficult, leading to a lack of consensus over many basic questions regarding the mechanisms involved, the oligomerization state of the enzyme in vivo and even the exact biochemical composition.This review is a summary of the progress towards the production of a structural model of PS II-derived from either X-ray crystallography or electron microscopy based techniques-and the current opinions, which have arisen from these structural analyses, on the structural topology and assemblage of the various subunits that constitute the complex.Abbreviations C₁₂-M dodecyl maltoside - CP chlorophyll protein - cyt b-559 cytochrome b-559 - DMPC dimyristoyl phosphatidyl choline - EC electron crystallography - EM electron microscopy - LHC II light harvesting complex II - OEC oxygen evolving complex - OG octyl--glucopyranoside - PS I Photosystem I - PS II Photosystem II - Tris N-tris (hydroxymethyl) amino ethane     

Electron microscopy in structural studies of Photosystem II

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment–protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II. This revised version was published online in August 2006 with corrections to the Cover Date.     

A three-dimensional study of the normal human placental villous core

Summary In order to study the three-dimensional ultrastructure of the Hofbauer cells, human placentae from the 6th to the 21 st week of gestation and also from the end of pregnancy were cryofractured and observed by scanning electron microscopy. Hofbauer cells were found in the villous core at all the gestation stages examined. Their surface morphology was characterized by lamellipodia, funnel-like structures, blebs and microplicae. This pleomorphic aspect was probably related to functional or environmental conditions. In addition, thin cytoplasmic processes connected the Hofbauer cells with each other and with the components of the villous stroma. Fractured Hofbauer cells revealed large vacuoles in the cytoplasm; the vacuoles were smaller in size both at the beginning and at the end of pregnancy. This study further attests to the macrophagic nature of these cells.     

Resonance Raman spectroscopy of carotenoids in Photosystem II core complexes

Resonance Raman (RR) spectroscopy has been used to examine the configuration of the carotenoids bound to Synechocystis PCC 6803 Photosystem II (PS II) core complexes. The excitation wavelengths used (514.5, 488.0, 476.5 and 457.9 nm) span the absorption bands of all of the ~12–17 neutral carotenoids in the PS II core complex. The RR spectra of the two carotenoids associated with the D1–D2 polypeptides (Car₅₀₇ and Car₄₈₉) of the reaction center are extracted via light versus dark difference experiments measured at 20 K. The RR results are consistent with all-trans configurations for both Car₅₀₇ and Car₄₈₉ and indicate that majority of the other carotenoids in the PS II core complex must also be in the all-trans configuration. The configuration of β-carotene is relevant to its proposed function as a molecular wire in the secondary electron-transfer reactions of PS II.     

Photosystem I from the unusual cyanobacterium Gloeobacter violaceus

Photosystem I (PS I) from the primitive cyanobacterium Gloeobacter violaceus has been purified and characterised. Despite the fact that the isolated complexes have the same subunit composition as complexes from other cyanobacteria, the amplitude of flash-induced absorption difference spectra indicates a much bigger antenna size with about 150 chlorophylls per P700 as opposed to the usual 90. Image analysis of the PS I preparation from Gloeobacter reveals that the PS I particles exist both in a trimeric and in a monomeric form and that their size and shape closely resembles other cyanobacterial PS I particles. However, the complexes exhibit a higher molecular weight as could be shown by gel filtration. The preparation contains novel polypeptides not related to known Photosystem I subunits. The N-terminal sequence of one of those polypeptides has been determined and reveals no homology to known or hypothetical proteins. Immunoblotting shows a cross-reaction of three of the polypeptide bands with an antibody raised against the major LHC from the diatom Cyclotella cryptica. Electron microscopy reveals a novel T-shaped complex which has never been observed in any other cyanobacterial PS I preparation. 77 K spectra of purified PS I show an extreme blue-shift of the fluorescence emission, indicating an unusual organisation of the PS I antenna system in Gloeobacter. This revised version was published online in June 2006 with corrections to the Cover Date.     

Three-Dimensional Electron Microscopy of the Photosystem II Core Complex

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochromeb-559, andpsbI gene product was reconstructed at 20-Å resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had ap22₁2₁symmetry. One PSII core complex was measured to be 80 × 80 Å in the membrane plane and 88 Å normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

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-α,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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.     

The three-dimensional arcitecture of the EJC core

The exon junction complex (EJC) is a macromolecular complex deposited at splice junctions on mRNAs as a consequence of splicing. At the core of the EJC are four proteins: eIF4AIII, a member of the DExH/D-box family of NTP-dependent RNA binding proteins, Y14, Magoh, and MLN51. These proteins form a stable heterotetramer that remains bound to the mRNA throughout many different cellular environments. We have determined the three-dimensional (3D) structure of this EJC core using negative-stain random-conical tilt electron microscopy. This structure represents the first structure of a DExH/D-box protein in complex with its binding partners. The EJC core is a four-lobed complex with a central channel and dimensions consistent with its known RNA footprint of about ten nucleotides. Using known X-ray crystallographic structures and a model of three of the four components, we propose a model for complex assembly on RNA and explain how Y14:Magoh may influence eIF4AIII's RNA binding.     

Excitation energy transfer and charge separation in the isolated Photosystem II reaction center

The nature of excitation energy transfer and charge separation in isolated Photosystem II reaction centers is an area of considerable interest and controversy. Excitation energy transfer from accessory chlorophyll a to the primary electron donor P680 takes place in tens of picoseconds, although there is some evidence that thermal equilibration of the excitation between P680 and a subset of the accessory chlorophyll a occurs on a 100-fs timescale. The intrinsic rate for charge separation at low temperature is accepted to be ca. (2 ps)^–1, and is based on several measurements using different experimental techniques. This rate is in good agreement with estimates based on larger sized particles, and is similar to the rate observed with bacterial reaction centers. However, near room temperature there is considerable disagreement as to the observed rate for charge separation, with several experiments pointing to a ca. (3 ps)^–1 rate, and others to a ca. (20 ps)^-1 rate. These processes and the experiments used to measure them will be reviewed.Abbreviations Chl chlorophyll - FWHM full-width at half-maximum - Pheo pheophytin - PS II Photosystem II - P680 primary electron donor of the Photosystem II reaction center - RC reaction center The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Associations between light-harvesting complexes and Photosystem II from Marchantia polymorpha L. determined by two- and three-dimensional electron microscopy

Assemblies of Photosystem II and light-harvesting proteins were purified from the liverwort Marchantia polymorpha and investigated by two- and three-dimensional transmission electron microscopy of negatively stained specimens. By single-particle analysis, it was determined that about 25% of the particles are rectangular or slightly S-shaped with dimensions of 285 A in length, 144 A in width, 84 A in height, while the membrane part is about 52 A thick. This structure reveals the same architecture as that of a Photosystem II-light-harvesting assembly from seed plants. An overlay of the projection structure of the liverwort's complex with a projection structure deduced from stained trimeric LHC II crystals from pea confirmed the locations of trimeric LHC II within the liverwort's complex. Remarkably tight associations of LHC II and other chlorophyll a/b binding proteins with the PS II core complex are observed. More than 50% of the Photosystem II particles from the liverwort carry one or two additional masses. These extra masses are found to consist of an additional LHC II trimer and probably a chlorophyll a/b binding protein. For the first time, a three-dimensional structure of such a large assembly is defined.     

A complementary microscopy analysis of Sticholysin II crystals on lipid films: Atomic force and transmission electron characterizations

A new crystal form of the cytotoxin Sticholysin II (StnII) formed on lipid monolayers of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) has been characterized by transmission electron microscopy (TEM) and by tapping mode atomic force microscopy (AFM) under nearly physiological conditions. Both approaches show the existence of single- and double-layered 2D crystals possessing hexagonal symmetry and unit cell dimensions of a = b =10 nm and gamma = 120 degrees. However, single-layered StnII crystals could only be analysed by TEM and double-layered crystals by AFM. Considering the previously known atomic structure of native StnII and that of a tetrameric assembly, a model is proposed for this new crystal form in which StnII conserves its monomeric state upon interaction with the lipid monolayer. These results are in agreement with the existence of the so called M2 state of the actinoporins.     

The major light-harvesting complex of Photosystem II: aspects of its molecular and cell biology

The light-harvesting complex of photosystem II (LHC II) contains one major (LHC IIb) and at least three minor chlorophyll-protein components. The apoproteins of LHC IIb (LHCP) are encoded by nuclear genes and synthesized in the cytoplasm as a higher molecular weight precursor(s) (pLHCP). Several genes coding for pLHCP have been cloned from various higher plant species. The expression of these genes is dependent upon a variety of factors such as light, the developmental stage of the plastids and the plant. After its synthesis in the cytoplasm, pLHCP is imported into plastids, inserted into thylakoids, processed to its mature form, and assembled into LHC IIb. The pathway of assembly of LHC IIb in the thylakoid membranes is currently being investigated in several laboratories. We present a model that gives some details of the steps in the assembly process. Many of the steps involved in the synthesis and assembly are dependent on light and the stage of plastid development.Abbreviations PS Photosystem - LHC II Light-harvesting complex of PS II - LHCP Apoproteins of LHC IIb - pLHCP Precursor of LHCP - PAGE Polyacrylamide gel electrophoresis     

Interaction of Photosystem 2-LHC2 Supercomplexes in Adjacent Layers of Stacked Chloroplast Thylakoid Membranes

Two Types of photosystem 2-light-harvesting complex 2 (PS2-LHC2) supercomplexes with similar pigment and protein composition were isolated directly from thylakoid membranes by sucrose density gradient centrifugation. Electron microscopy and single particle image analysis revealed the first Type as single unpaired PS2-LHC2 supercomplexes, whereas the second Type was characterized as pairs of two PS2-LHC2 supercomplexes attached together by their stromal sides. Unstacking of thylakoid membranes resulted in a spontaneous disintegration of the paired supercomplexes into single unpaired particles. A model of the organisation of the pigment-protein complexes in grana region is proposed.     

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Polypeptide composition of a Photosystem II core complex. Presence of a herbicide-binding protein

The polypeptide composition of a Photosystem II (PS II) core complex from higher plant chloroplasts has been characterized by subjecting the isolated complex to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides in the 40–50 kDa size class, attributed to the chlorophyll a-binding apoproteins of PS II, were resolved when the urea concentration in the SDS-polyacrylamide gel electrophoresis was greater than 1 M. The two chlorophyll a-binding proteins were dissimilar in their primary structure based upon their different hydrolysis products on SDS-polyacrylamide gel electrophoresis following papain treatment. The core complex contained three additional polypeptides. Two polypeptides in the 30–34 kDa size class were resolved when the urea concentration in the gel system was increased to greater than 4 M. One of the polypeptides in this size class was identified as the herbicide-binding protein from azido[¹⁴C]atrazine labeling studies. The herbicide-binding protein displayed an anomalous electrophoretic migration behavior in SDS-polyacrylamide gel electrophoresis in the presence or absence of urea; its apparent molecular weight decreased when the urea concentration increased. The fifth protein component of the core complex was attributed to cytochrome b-559 which was found to consist of the ascorbate- and dithionite-reducible forms in the samples prior to SDS solubilization.     

Structure of the mitochondrial outer membrane channel derived from electron microscopy of 2D crystals

A structural model for the channel in the mitochondrial outer membrane is presented, derived from electron microscopic studies of two-dimensional crystals and inferences from the primary structure of the 30-kDa polypeptide which forms the channel. The channel is represented as a cylindrical beta-barrel, with a carbon backbone diameter of 3.8 nm. The axial projection of the cylinder is divided radially into four sectors by four interchannel contact points. These sectors are characterized in terms of their interactions with lipid and macromolecular ligands, and in terms of the presence or absence of exposed basic amino acids.     

A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions

The structure and polypeptide composition of the photosynthetic membrane of a mutant of maize has been investigated. The thylakoid membranes of the mutant plants are deficient in Photosystem I activity, although Photosystem II is at near normal levels. SDS polyacrylamide gel electrophoresis of thylakoid membranes from the mutant shows them to be deficient in two polypeptide bands which have been associated with Photosystem I. Freeze-fracture studies of the membrane show that the absence of these polypeptides is associated with a measurable reduction in particle diameter on the unstacked protoplasmic fracture face. This fracture face is derived from the splitting of membranes in unstacked regions of the thylakoid membrane system. It is suggested that in membranes stacked by salts in vitro, Photosystem I activity may be confined to this region.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.