Insecticidal toxins from black widow spider venom.

The biological effects of Latrodectus spider venom are similar in animals from different phyla, but these symptoms are caused by distinct phylum-specific neurotoxins (collectively called latrotoxins) with molecular masses ranging from 110 to 140 kDa. To date, the venom has been found to contain five insecticidal toxins, termed alpha, beta, gamma, delta and epsilon-latroinsectotoxins (LITs). There is also a vertebrate-specific neurotoxin, alpha-latrotoxin (alpha-LTX), and one toxin affecting crustaceans, alpha-latrocrustatoxin (alpha-LCT). These toxins stimulate massive release of neurotransmitters from nerve terminals and act (1) by binding to specific receptors, some of which mediate an exocytotic signal, and (2) by inserting themselves into the membrane and forming ion-permeable pores. Specific receptors for LITs have yet to be identified, but all three classes of vertebrate receptors known to bind alpha-LTX are also present in insects. All LTXs whose structures have been elucidated (alpha-LIT, delta-LIT, alpha-LTX and alpha-LCT) are highly homologous and have a similar domain architecture, which consists of a unique N-terminal sequence and a large domain composed of 13-22 ankyrin repeats. Three-dimensional (3D) structure analysis, so far done for alpha-LTX only, has revealed its dimeric nature and an ability to form symmetrical tetramers, a feature probably common to all LTXs. Only tetramers have been observed to insert into membranes and form pores. A preliminary 3D reconstruction of a delta-LIT monomer demonstrates the spatial similarity of this toxin to the monomer of alpha-LTX.

Low-light-adapted Prochlorococcus species possess specific antennae for each photosystem.

Prochlorococcus, the most abundant genus of photosynthetic organisms, owes its remarkably large depth distribution in the oceans to the occurrence of distinct genotypes adapted to either low- or high-light niches. The pcb genes, encoding the major chlorophyll-binding, light-harvesting antenna proteins in this genus, are present in multiple copies in low-light strains but as a single copy in high-light strains. The basis of this differentiation, however, has remained obscure. Here we show that the moderate low-light-adapted strain Prochlorococcus sp. MIT 9313 has one iron-stress-induced pcb gene encoding an antenna protein serving photosystem I (PSI)--comparable to isiA genes from cyanobacteria--and a constitutively expressed pcb gene encoding a photosystem II (PSII) antenna protein. By comparison, the very low-light-adapted strain SS120 has seven pcb genes encoding constitutive PSI and PSII antennae, plus one PSI iron-regulated pcb gene, whereas the high-light-adapted strain MED4 has only a constitutive PSII antenna. Thus, it seems that the adaptation of Prochlorococcus to low light environments has triggered a multiplication and specialization of Pcb proteins comparable to that found for Cab proteins in plants and green algae.

Organization of transmembrane helices in photosystem II: comparison of plants and cyanobacteria.

Electron microscopy and X-ray crystallography are revealing the structure of photosystem II. Electron crystallography has yielded a 3D structure at sufficient resolution to identify subunit positioning and transmembrane organization of the reaction-centre core complex of spinach. Single-particle analyses are providing 3D structures of photosystem II-light-harvesting complex II supercomplexes that can be used to incorporate high-resolution structural data emerging from electron and X-ray crystallography. The positions of the chlorins and metal centres within photosystem II are now available. It can be concluded that photosystem II is a dimeric complex with the transmembrane helices of CP47/D2 proteins related to those of the CP43/D1 proteins by a twofold axis within each monomer. Further, both electron microscopy and X-ray analyses show that P(680) is not a 'special pair' and that cytochrome b559 is located on the D2 side of the reaction centres some distance from P(680). However, although comparison of the electron microscopy and X-ray models for spinach and Synechococcus elongatus show considerable similarities, there seem to be differences in the number and positioning of some small subunits.

Three-dimensional structure of the photosystem II core dimer of higher plants determined by electron microscopy.

Here we report the first three-dimensional structure of a higher plant photosystem II core dimer determined by electron crystallography at a resolution sufficient to assign the organization of its transmembrane helices. The locations of 34 transmembrane helices in each half of the dimer have been deduced, 22 of which are assigned to the major subunits D1 (5), D2 (5), CP47 (6), and CP43 (6). CP47 and CP43, located on opposite sides of the D1/D2 heterodimer, are structurally similar to each other, consisting of 3 pairs of transmembrane helices arranged in a ring. Both CP47 and CP43 have densities protruding from the lumenal surface, which are assigned to the loops joining helices 5 and 6 of each protein. The remaining 12 helices within each half of the dimer are attributed to low-molecular-weight proteins having single transmembrane helices. Comparison of the subunit organization of the higher plant photosystem II core dimer reported here with that of its thermophilic cyanobacterial counterpart recently determined by X-ray crystallography shows significant similarities, indicative of a common evolutionary origin. Some differences are, however, observed, and these may relate to variations between the two classes of organisms in antenna linkage or thermostability.

Oxyphotobacteria. Antenna ring around photosystem I.

The oceanic picoplankton Prochlorococcus - probably the most abundant photosynthetic organism on our planet - can grow at great depths where light intensity is very low. We have found that the chlorophyll-binding proteins in a deep-living strain of this oxyphotobacterium form a ring around a trimer of the photosystem I (PS I) photosynthetic reaction centre, a clever arrangement that maximizes the capture of light energy in such dim conditions.

Subunit positioning and transmembrane helix organisation in the core dimer of photosystem II.

Recently 3D structural models of the photosystem II (PSII) core dimer complexes of higher plants (spinach) and cyanobacteria (Synechococcus elongatus) have been derived by electron [Rhee et al. (1998) Nature 396, 283-286; Hankamer et al. (2001) J. Struct. Biol., in press] and X-ray [Zouni et al. (2001) Nature 409, 739-743] crystallography respectively. The intermediate resolutions of these structures do not allow direct identification of side chains and therefore many of the individual subunits within the structure are unassigned. Here we review the structure of the higher plant PSII core dimer and provide evidence for the tentative assignment of the low molecular weight subunits. In so doing we highlight the similarities and differences between the higher plant and cyanobacterial structures.

Three-dimensional model and characterization of the iron stress-induced CP43'-photosystem I supercomplex isolated from the cyanobacterium Synechocystis PCC 6803.

The cyanobacterium Synechocystis PCC 6803 has been subjected to growth under iron-deficient conditions. As a consequence, the isiA gene is expressed, and its product, the chlorophyll a-binding protein CP43', accumulates in the cell. Recently, we have shown for the first time that 18 copies of this photosystem II (PSII)-like chlorophyll a-binding protein forms a ring around the trimeric photosystem I (PSI) reaction center (Bibby, T. S., Nield, J., and Barber, J. (2001) Nature, 412, 743-745). Here we further characterize the biochemical and structural properties of this novel CP43'-PSI supercomplex confirming that it is a functional unit of approximately 1900 kDa where the antenna size of PSI is increased by 70% or more. Using electron microscopy and single particle analysis, we have constructed a preliminary three-dimensional model of the CP43'-PSI supercomplex and used it as a framework to incorporate higher resolution structures of PSI and CP43 recently derived from x-ray crystallography. Not only does this work emphasize the flexibility of cyanobacterial light-harvesting systems in response to the lowering of phycobilisome and PSI levels under iron-deficient conditions, but it also has implications for understanding the organization of the related chlorophyll a/b-binding Pcb proteins of oxychlorobacteria, formerly known as prochlorophytes.

Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria.

Although iron is the fourth most abundant element in the Earth's crust, its concentration in the aquatic ecosystems-particularly the open oceans-is sufficiently low to limit photosynthetic activity and phytoplankton growth. Cyanobacteria, a major class of phytoplankton, respond to iron deficiency by expressing the 'iron-stress-induced' gene, isiA(ref. 3). The protein encoded by this gene has an amino-acid sequence that shows significant homology with one of the chlorophyll a-binding proteins (CP43) of photosystem II (PSII). The precise function of the CP43-like protein, here called CP43', has not been elucidated, although there have been many suggestions. Here we show that CP43' associates with photosystem I (PSI) to form a complex that consists of a ring of 18 CP43' molecules around a PSI trimer. This significantly increases the size of the light-harvesting system of PSI. The utilization of a PSII-like protein as an extra antenna for PSI emphasises the flexibility of cyanobacterial light-harvesting systems, and seems to be a strategy which compensates for the lowering of phycobilisome and PSI levels in response to iron deficiency.

Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D reconstruction.

Single particle analysis and 3D reconstruction of molecules imaged by transmission electron microscopy have provided a wealth of medium to low resolution structures of biological molecules and macromolecular complexes, such as the ribosome, viruses, molecular chaperones and photosystem II. In this review, the principles of these techniques are introduced in a non-mathematical way, and single particle analysis is compared to other methods used for structural studies. In particular, the recent X-ray structures of the ribosome and of ribosomal subunits allow a critical comparison of single particle analysis and X-ray crystallography. This has emphasised the rapidity with which single particle analysis can produce medium resolution structures of complexes that are difficult to crystallise. Once crystals are available, X-ray crystallography can produce structures at a much higher resolution. The great similarities now seen between the structures obtained by the two techniques reinforce confidence in the use of single particle analysis and 3D reconstruction, and show that for electron cryo-microscopy structure distortion during sample preparation and imaging has not been a significant problem. The ability to analyse conformational flexibility and the ease with which time-resolved studies can be performed are significant advantages for single particle analysis. Future improvements in single particle analysis and electron microscopy should increase the attainable resolution. Combining single particle analysis of macromolecular complexes and electron tomography of subcellular structures with high-resolution X-ray structures may enable us to realise the ultimate dream of structural biology-a complete description of the macromolecular complexes of the cell in their different functional states.

Supermolecular structure of photosystem II and location of the PsbS protein.

This paper addresses the question of whether the PsbS protein of photosystem two (PS II) is located within the LHC II PS II supercomplex for which a three-dimensional structure has been obtained by cryoelectron microscopy and single particle analysis. The PsbS protein has recently been implicated as the site for non-photochemical quenching. Based both on immunoblotting analyses and structural considerations of an improved model of the spinach LHC II PS II supercomplex, we conclude that the PsbS protein is not located within the supercomplex. Analyses of other fractions resulting from the solubilization of PS Il-enriched membranes derived from spinach suggest that the PsbS protein is located in the LHC II-rich regions that interconnect the supercomplex within the membrane.

Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization.

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.

3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis.

Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.

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.

Self-incompatibility in the grasses: evolutionary relationship of the S gene from Phalaris coerulescens to homologous sequences in other grasses.

Self-incompatibility is widespread in the grasses and it is proposed that the grasses share a common incompatibility mechanism that is distinct from those operating in the dicotyledonous species studied in great detail. Where good genetic data are available, all grass species appear to have an incompatibility mechanism controlled by two unlinked loci, S and Z. A putative S gene has been cloned from Phalaris coerulescens. This gene is characterized by two major domains: an allele specificity domain and a thioredoxin catalytic domain. A family of sequences with varying degrees of homology to this gene has been identified among 15 grass species covering all subfamilies of the Poaceae. These S-related sequences appear to be present in the grass family regardless of self-compatibility. Evidence is presented to show that at least one of the sequences is transcribed, suggesting a functional gene. In contrast to the high expression of the S gene in Phalaris pollen, expression of the related gene in the pollen (or anthers) of the grass species examined was so low that RNA gel blot analysis failed to display a significant signal. However, reverse transcription-based polymerase chain reaction (RT-PCR) successfully amplified the region corresponding to the S thioredoxin domain from 10 of the grass species. With grasses other than Phalaris, RT-PCR showed limited success in amplifying the region corresponding to the S variable portion at the 5' end of the Phalaris S gene. Sequencing of the PCR-amplified S thioredoxin region from wheat, barley, rye and Dactylis revealed that this is a highly conserved gene with 94-97% sequence similarity with the corresponding Phalaris S gene. The conservation of sequence and ubiquitous expression of the gene across the grass family strongly suggest that the S-related gene is carrying out a significant biological function in the Poaceae. On the basis of these findings, a model for the evolution of the S self-incompatibility gene in the grasses is proposed.

Isolation and biochemical characterisation of monomeric and dimeric photosystem II complexes from spinach and their relevance to the organisation of photosystem II in vivo.

Membranes enriched in photosystem II were isolated from spinach and further solubilised using n-octyl beta-D-glucopyranoside (OctGlc) and n-dodecyl beta-D-maltoside (DodGlc2). The OctGlc preparation had high rates of oxygen evolution and when subjected to size-exclusion HPLC and sucrose density gradient centrifugation, in the presence of DodGlc2, separated into dimeric (430 kDa), monomeric (236 kDa) photosystem II cores and a fraction containing photosystem II light-harvesting complex (Lhcb) proteins. The dimeric core fraction was more stable, contained higher levels of chlorophyll, beta-carotene and plastoquinone per photosystem II reaction centre and had a higher oxygen-evolving activity than the monomeric cores. Their subunit composition was similar (CP43, CP47, D1, D2, cytochrome b 559 and several lower-molecular-mass components) except that the level of 33-kDa extrinsic protein was lower in the monomeric fraction. Direct solubilisation of photosystem-II-enriched membranes with DodGlc2, followed by sucrose density gradient centrifugation, yielded a super complex (700 kDa) containing the dimeric form of the photosystem II core and Lhcb proteins: Lhcb1, Lhcb2, Lhcb4 (CP29), and Lhcb5 (CP26). Like the dimeric and monomeric photosystem II core complexes, the photosystem II-LHCII complex had lost the 23-kDa and 17-kDa extrinsic proteins, but maintained the 33-kDa protein and the ability to evolve oxygen. It is suggested, with a proposed model, that the isolated photosystem II-LHCII super complex represents an in vivo organisation that can sometimes form a lattice in granal membranes of the type detected by freeze-etch electron microscopy [Seibert, M., DeWit, M. & Staehelin, L. A. (1987) J. Cell Biol. 105, 2257-2265].

A self-fertile mutant of Phalaris produces an S protein with reduced thioredoxin activity.

Gametophytic self-incompatibility in the Phalaris coerulescens is controlled by two unlinked genes, S and Z. Isolation of the S gene from the pollen of this grass species indicated that the C terminus has significant homology with thioredoxin H proteins. The protein from the C terminus, expressed in Escherichia coli, exhibits thioredoxin-life activity. This paper demonstrates that the C terminus of the S protein from an S complete mutant shows significant reduction in thioredoxin activity when compared with the wild-type form. Both pollen and stigma have lost self-compatibility in this mutant. Close examination of the lesions, which were found only in the C terminus of the mutant gene suggests that the substitution of a serine by an arginine is responsible for the reduced enzymatic activity. The association between reduced activity and the loss of the self-incompatibility provides evidence for a role of thioredoxin activity in the self-incompatibility reaction of this species.

Thioredoxin activity in the C terminus of Phalaris S protein.

Self-incompatibility in the grass Phalaris coerulescens is controlled by two genes S and Z. Isolation and sequencing of two S alleles showed that they encode proteins that are highly conserved at the C terminus with significant homology to thioredoxin H proteins. In particular, the residues in and around the active site of thioredoxin, Trp-Cys-Gly-Pro-Cys, are perfectly conserved. The C terminus of the S protein has been expressed in Eschericia coli and purified to homogeneity on Ni-NTA resin. Functional assays showed that the protein has thioredoxin activity; it can act as a substrate for E. coli thioredoxin reductase and also catalyse the reduction of insulin by dithiothreitol. The possible role of thioredoxin-like activity of the S protein in mediating the incompatibility reaction in Phalaris is discussed.

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.

Comparative costs of lung cancer management.

A method is outlined for comparative costing of different protocols of lung cancer management in a free-standing clinic. The costs of chemotherapy and radiation therapy are evaluated according to alternative regimens of treatment. The costs of new patient assessment, patient follow-up, and ancillary care (social work and nutrition assistance) are also included. Except for the cost of routine blood tests during chemotherapy and radiotherapy planning computerized tomography (CT) scans, costs incurred outside the clinic are excluded. The method is illustrated by application to out-patient treatment of lung cancer at the Victoria Clinic of the British Columbia Cancer Agency in Victoria, British Columbia, Canada, using data for the 1989-90 fiscal year of the Clinic. The method may be adapted for use in other disease and institutional settings.