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Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z ring stabilization is not well-understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission EM. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly.

Cd(2+) and Pb(2+) are xenobiotic heavy metal ions that use ionic mimicry to interfere with the cellular function of biomacromolecules. Using a combination of SAXS, electron microscopy, FRET, and solution NMR spectroscopy, we demonstrate that treatment with Cd(2+) and Pb(2+) causes self-assembly of protein kinase C regulatory domains that peripherally associate with membranes. The self-assembly process successfully competes with ionic mimicry and is mediated by conserved protein regions that are distinct from the canonical Ca(2+)-binding motifs of protein kinase C. The ability of protein oligomers to interact with anionic membranes is enhanced compared to the monomeric species. Our findings suggest that metal-ion-dependent peripheral membrane domains can be utilized for generating protein–metal-ion nanoclusters and serve as biotemplates for the design of sequestration agents.

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.

Chloroplast division requires filamentation temperature-sensitive Z (FtsZ), a tubulin-like GTPase of cyanobacterial endosymbiotic origin. Plants and algae possess two distinct FtsZ protein families, FtsZ1 and FtsZ2 that co-assemble into a ring (Z-ring) at the division site. Z-ring assembly and disassembly and division site positioning is controlled by both positive and negative factors via their specific interactions with FtsZ1 and FtsZ2. Here we present thein plantaanalysis ofArabidopsisFtsZ1 and FtsZ2 turnover in the context of a native chloroplast division machinery. Fluorescence recovery after photobleaching analysis was conducted using fluorescently tagged FtsZ at wild-type (WT)-like levels. Rapid photobleaching, low signal-to-noise ratio, and phototropic movements of chloroplasts were overcome by (i) using progressive intervals in time-lapse imaging, (ii) analyzing epidermal rather than stromal chloroplasts, and (iii) employing image stack alignment during postprocessing. In plants of WT background, fluorescence recovery half-times averaged 117 and 325 s for FtsZ1 and FtsZ2, respectively. In plants lacking ARC3, the key negative regulator of FtsZ assembly, the turnover was threefold slower. The findings are discussed in the context of previous results conducted in a heterologous system.

The enzyme-linked immunosorbent assay (ELISA) constitutes an important clinical diagnostic approach. However, the prolonged incubation times involved lead to turnaround times of typically ⩾1 day, potentially delaying a definitive diagnosis or an adequate treatment plan for individual patients. Here cold-microwave technology (CMT) was employed to significantly reduce the times required for diagnostic ELISAs. The new approach was validated and compared to a conventional ELISA setup measuring canine calprotectin (cCP). Canine serum and fecal specimens were used for the analytical validation of cCP ELISA by conventional and CMT–ELISA. Cross-validation of both ELISA methods consisted of the determination of analytic sensitivity, linearity, accuracy, precision, and reproducibility. The long-term stability of antibody-coated ELISA plates was also evaluated up to 33 days. The ELISA approaches were comparable to each other. The observed-to-expected ratios for linearity and accuracy were 100.2 ± 11.8 and 98.1 ± 10.8% (mean ± standard deviation), respectively. Precision and reproducibility were ⩽17.2%. For samples run on precoated ELISA plates over 33 days %CVs were ⩽12.5%. While both ELISA approaches were analytically sensitive, linear, accurate, precise, and reproducible with measurements of cCP concentrations, CMT–ELISA offered a reduction in incubation times by 90–95%, facilitating a very fast turnaround time and suggesting CMT–ELISA for improved human and veterinary clinical diagnostics.

Small molecule probes for perturbing protein-protein interactions (PPIs) in vitro can be useful if they cause the target proteins to undergo biomedically relevant changes to their tertiary and quaternary structures. Application of the Exploring Key Orientations (EKO) strategy (J. Am. Chem. Soc., 2013, 135, 167 – 173) to a piperidinone-piperidine chemotype 1 indicated specific derivatives were candidates to perturb a protein-protein interface in the α-antithrombin dimer; those particular derivatives of 1 were prepared and tested. In the event, most of them significantly accelerated oligomerization of monomeric α-antithrombin, which is metastable in its oligomeric state. This assertion is supported by data from gel electrophoresis (non-denaturing PAGE; throughout) and probe-induced loss of α-antithrombin’s inhibitor activity in a reaction catalyzed by thrombin. Kinetics of α-antithrombin oligomerization induced by the target compounds were examined. It was found that probes with O-benzyl-protected serine side-chains are the most active catalysts in the series, and reasons for this, based on modeling experiments, are proposed. Overall, this study reveals one of the first examples of small molecules designed to act at a protein-protein interface relevant to oligomerization of a serpin (ie α-antithrombin). The relevance of this to formation of oligomeric serpin fibrils, associated with the disease states known as “serpinopathies”, is discussed.

At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S(21) 68. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.

Plant and algal chloroplasts and their ultrastructure were one of the first specimens to be studied by microscopy, and their granular nature was first described by Van Leeuwenhoek in 1676. Here we have used cryo-electron tomography and three-dimensional reconstruction to resolve chloroplast ultrastructure at a level where individual protein complexes can be observed. The data reveal features of the ultrastructure which are consistent with a simple hypothesis for the generation of the characteristic stacked grana described by Leeuwenhoek. We find that stacked regions can be formed by invaginations of the ‘sac-like’ structure of the membrane. Two, or more, invaginations associate together to give stacks of membranes within the sac. In the tomograms, both the stacked and unstacked regions of the membrane display lateral heterogeneity in terms of the particles embedded in them. Unstacked regions display microdomains that are densely packed with CF1-CF0 proton ATPases that predominantly occupy parts where the disk-shaped sacs curve at the periphery. Stacked regions display membranes packed with photosystem II complexes.

BACKGROUND: The ultrastructural characterisation of cellular components is a key element in revealing the bases for differences in nutrient bioaccessibility among fruits and vegetables and their derived products. Together, cell walls and chromoplasts constitute the two major physical barriers to carotenoid release from the food matrix (structure) during digestion. In general, larger cells with thinner cell walls are most likely to fail under mechanical pressure. In relation to chromoplasts, the substructures plastoglobuli, crystals and membranes give decreasing rates of carotenoid solubilisation when exposed to digestive forces. RESULTS: This paper describes cell wall and chromoplast structures in nine carotenoid-storing raw fruits and vegetables. Watermelon and melon cells were shown to have the largest cells concomitant with thin, non-fibrous cell walls, while carrot, hypodermal grapefruit and sweet potato cells were smallest with fibrous or dense cell walls. Mango fruit showed the highest proportion of globules to other substructures. Carrot, papaya and tomato contained many crystalline structures. Finally, watermelon, mango and butternut squash developed a high proportion of membranous structures. CONCLUSION: A more precise description of the physical characteristics of foods that stand as barriers to bioaccessibility can help in understanding which are more or less inhibitory for particular foods. Copyright © 2012 Society of Chemical Industry

Protein Kinase C (PKC) isoenzymes are a family of kinases that control cell proliferation, migration, and apoptosis. The hallmark of PKC activation is their translocation to lipid membranes that occurs in response to Ca2+ and diacylglycerol. Previous studies have shown that PKC activity is modulated by toxic metal ions such as Pb2+ and Cd2+. Our objective was to understand the effect of these metal ions on the structure and function of the membrane-binding regulatory region of PKC. Our efforts focused on the PKCα region that consists of two peripheral membrane-binding domains, C1B and C2. Using a combination of biophysical techniques, we demonstrate that interactions of Pb2+ and Cd2+ with C1B-C2 have profound and distinct consequences for the biophysical and functional properties of this region. While both Cd2+ and Pb2+ can spontaneously replace structural Zn2+ ions in C1B, NMR spectroscopy revealed that only Cd2+ was able to support the proper fold and ligand-binding function of the protein. In addition, Cd2+ promotes the formation of well-defined C1B-C2 oligomers that we were able to detect and characterize using small-angle X-ray scattering experiments and negative-stain electron microscopy. In contrast to Cd2+, Pb2+ cannot support the proper fold of C1B but acts successfully as a functional surrogate of Ca2+ in driving protein-membrane association. Our work demonstrates the potential diversity of responses of signaling proteins to toxic metal ions and suggests that molecular mechanisms of Pb2+ and Cd2+ toxicity are distinct. This work was supported by Welch Foundation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 GM108998.

In eukaryotes, calcium-binding proteins play a pivotal role in diverse cellular processes, and recent findings suggest similar roles for bacterial proteins at different stages in their life cycle. Here, we report the crystal structure of calcium dodecin, Rv0379, from Mycobacterium tuberculosis with a dodecameric oligomeric assembly and a unique calcium-binding motif. Structure and sequence analysis were used to identify orthologs of Rv0379 with different ligand-binding specificity.

Phage holins and endolysins have long been known to play key roles in lysis of the host cell, disrupting the cytoplasmic membrane and peptidoglycan (PG) layer, respectively. For phages of Gram-negative hosts, a third class of proteins, the spanins, are involved in disrupting the outer membrane (OM). Rz and Rz1, the components of the lambda spanin, are, respectively, a class II inner membrane protein and an OM lipoprotein, are thought to span the entire periplasm by virtue of C-terminal interactions of their soluble domains. Here, the periplasmic domains of Rz and Rz1 have been purified and shown to form dimeric and monomeric species, respectively, in solution. Circular dichroism analysis indicates that Rz has significant alpha-helical character, but much less than predicted, whereas Rz1, which is 25% proline, is unstructured. Mixture of the two proteins leads to complex formation and an increase in secondary structure, especially alpha-helical content. Moreover, transmission electron-microscopy reveals that Rz–Rz1 complexes form large rod-shaped structures which, although heterogeneous, exhibit periodicities that may reflect coiled-coil bundling as well as a long dimension that matches the width of the periplasm. A model is proposed suggesting that the formation of such bundles depends on the removal of the PG and underlies the Rz–Rz1 dependent disruption of the OM.

Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or “holes.” In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.

Plants and algae contain the FtsZ1 and FtsZ2 protein families that perform specific, non-redundant functions in plastid division. In vitro studies of chloroplast division have been hampered by the lack of a suitable expression system. Here we report the expression and purification of FtsZ1-1 and FtsZ2-1 from Arabidopsis thaliana using a eukaryotic host. Specific GTPase activities were determined and found to be different for FtsZ1-1 vs. FtsZ2-1. The purified proteins readily assembled into previously unreported assembly products named type-I and -II filaments. In contrast to bacterial FtsZ, the Arabidopsis proteins do not form bundled sheets in the presence of Ca2+.

In order for human cytomegalovirus (HCMV) to replicate, concatemeric DNA has to be cleaved into unit-length genomes and packaged into preformed capsids. For packaging to take place and DNA to be translocated, a channel is required in the capsid. Viral capsid channels are generally formed by portal proteins. Here, we show by cross-linking, native gel electrophoresis of infected cells and gel permeation chromatography that the HCMV portal candidate protein pUL104 can form dimers and higher order multimers. Electron microscopy of purified monomeric pUL104 after 5 min incubation revealed that the protein had assembled into a multimeric form and that this form closely resembles complete portal assembly. This is the first study to show that pUL104 monomers have the ability to form portal complexes without additional viral proteins.

article i nfo Article history: Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix α-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix α-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.

Hybrids of Dendrobium nobile Lindl. have high potential to become a high-value pot plant, but detailed research to support the development of commercial production protocols was lacking. A 3 × 5 factorial experiment was conducted to investigate the effects of nutrient termination date (1 Aug., 1 Sept., or 1 Oct.) and nutrient reapplication time (at the beginning or in the middle of cooling, immediately after or 2 weeks after the completion of cooling, or no nutrient reapplication) on growth and flower development of Dendrobium Sea Mary ‘Snow King,’ a D. nobile hybrid. Interaction between nutrient termination date and reapplication time on growth and flowering was nonsignificant for all variables measured, and reapplication time had only a minor effect on leaves remaining. Regardless of nutrient reapplication time, delaying nutrient termination date resulted in improved growth and flowering. Nutrient termination on 1 Oct. resulted in taller plants with more nodes, leaves remaining, flowering nodes, and total flowers as well as fewer aborted flowers than an earlier termination date. Nutrient supply until 1 Oct. did not lead to differences in time required for anthesis but extended the time needed to reach full flowering by 1.5 d. The results suggest that flower development benefited more from the nutrients that were accumulated in mature pseudobulbs before nutrient termination rather than from those being taken from the reapplied fertilizers. Only lateral buds protruding 2 mm or more from the pseudobulb surface showed differentiated floral structures when examined histologically. The buds, excised 4 weeks after cooling treatments began, showed that nutrient termination on 1 Aug. resulted in larger flower primordia than those ended on 1 Oct., indicating an earlier or faster flower differentiation with earlier nutrient termination. No aerial shoot formation or reversion of reproductive to vegetative buds arose as a result of either late nutrient termination or resumption of nutrient application.

RNA recognition receptors are important for detection of and response to viral infections. RIG-I and MDA5 are cytoplasmic DEX(D/H) helicase proteins that can induce signaling in response to RNA ligands, including those from viral infections. LGP2, a homolog of RIG-I and MDA5 without the caspase recruitment domain required for signaling, plays an important role in modulating signaling by MDA5 and RIG-I, presumably through heterocomplex formation and/or by serving as a sink for RNAs. Here we demonstrate that LGP2 can be coexpressed with RIG-I to inhibit activation of the NF-κB reporter expression and that LGP2 protein produced in insect cells can bind both single- and double-stranded RNA (dsRNA), with higher affinity and cooperativity for dsRNA. Electron microscopy and image reconstruction were used to determine the shape of the LGP2 monomer in the absence of dsRNA and of the dimer complexed to a 27-bp dsRNA. LGP2 has striking structural similarity to the helicase domain of the superfamily 2 DNA helicase, Hef.

One of the major challenges facing structural biologists today is the determination of high-resolution 3D structures of membrane proteins. The requirement for detergent molecules to be present makes X-ray crystallography particularly difficult, coupled with the added problems of isolating sufficient (viable) protein samples at high enough concentrations to yield 3D crystals. One technique that enables structural determination with fewer constraints is electron crystallography of two-dimensional crystals, in which small amounts of membrane proteins can be studied in native form in lipid bilayers.

Ro ribonucleoproteins are a class of antigenic ribonucleoproteins associated with rheumatic autoimmune diseases like systemic lupus erythematosus and Sjögrens syndrome in humans. Ro ribonucleoproteins are mostly composed of the 60-kDa Ro protein and small cytoplasmic RNAs, called Y RNAs, of unknown function. In eukaryotes, where Ro has been found to associate with damaged or mutant RNAs, it has been suggested that Ro may play a role in RNA quality control. In the radiation-resistant bacterium Deinococcus radiodurans and some eukaryotes, Ro has also been implicated in cell survival following UV damage. Here we present the first high resolution structure of a prokaryotic Ro ortholog, Rsr from D. radiodurans. The structure has been solved to 1.9 A resolution and shows distinct differences when compared with the eukaryotic apo- and RNA-bound Ro structures. Rsr is composed of two domains: a helical RNA binding domain and a mixed "von Willebrand factor A-like" domain containing a divalent metal binding site. Although the individual domains of Rsr are similar to the eukaryotic Ro, significantly large differences are seen at the interface of the two domains. Since this interface communicates with the conserved central cavity of Ro, which is implicated in RNA binding, changes at this interface could potentially influence RNA binding by Ro. Although the apo-Rsr protein is monomeric, Rsr binds Y RNA to form multimers of approximately 12 molecules of a 1:1 Rsr-Y RNA complex. Rsr binds D. radiodurans Y RNA with low nanomolar affinity, comparable with previously characterized eukaryotic Ro orthologs.

This study concerns the self-assembly of virus-like particles (VLPs) composed of an icosahedral virus protein coat encapsulating a functionalized spherical nanoparticle core. The recent development of efficient methods for VLP self-assembly has opened the way to structural studies. Using electron microscopy with image reconstruction, the structures of several VLPs obtained from brome mosaic virus capsid proteins and gold nanoparticles were elucidated. Varying the gold core diameter provides control over the capsid structure. The number of subunits required for a complete capsid increases with the core diameter. The packaging efficiency is a function of the number of capsid protein subunits per gold nanoparticle. VLPs of varying diameters were found to resemble to three classes of viral particles found in cells ( T = 1, 2, and 3). As a consequence of their regularity, VLPs form three-dimensional crystals under the same conditions as the wild-type virus. The crystals represent a form of metallodielectric material that exhibits optical properties influenced by multipolar plasmonic coupling.

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio is highly beneficial in many research areas. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, Western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 24-45 min.

The most advanced low-cost organic photovoltaic cells have a quantum efficiency of ~10%. This is in stark contrast to plant/bacterial light-harvesting systems which offer quantum efficiencies close to unity. Of particular interest is the highly effective quantum coherence-enabled energy transfer (Fig. 1). Noting that quantum coherence is promoted by charged residues and local dielectrics, classical atomistic simulations and time-dependent density functional theory (DFT) are used to identify charge/dielectric patterns and electronic coupling at exactly defined energy transfer interfaces. The calculations make use of structural information obtained on photosynthetic protein-pigment complexes while still in the native membrane making it possible to establish a link between supramolecular organization and quantum coherence in terms of what length scales enable fast energy transport and prevent quenching. Calculating energy transfer efficiencies between components based on different proximities will permit the search for patterns that enable defining material properties suitable for advanced photovoltaics.

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues.1., 2., 3. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5′ and 3′ of the uridine.1., 2. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3′ of the recognition uridylate, with some cleavage 3′ of cytidylate. A highly conserved RNA structure in the 3′ non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn2+. The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 Å structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-kappaB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.

The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.

We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

GroEL can solubilize membrane proteins by binding them in its hydrophobic cavity when detergent is removed by dialysis. The best-studied example is bacteriorhodopsin, which can bind in the GroEL chaperonin at two molecules per tetradecamer. Applying this approach to the holin and antiholin proteins of phage lambda, we find that both proteins are solubilized by GroEL, in an ATP-sensitive mode, but to vastly different extents. The antiholin product, S107, saturates the chaperonin at six molecules per tetradecameric complex, whereas the holin, S105, which is missing the two N-terminal residues of S107, forms a hyper-solubilization complex with up to 350 holin molecules per GroEL, or approximately 4 MDa of protein per 0.8 MDa tetradecamer. Gel filtration chromatography and immunoprecipitation experiments confirmed the existence of complexes of the predicted masses for both S105 and S107 solubilization. For S105, negatively stained electron microscopic images show structures consistent with protein shells of the holin assembled around the chaperonin tetradecamer. Importantly, S105 can be delivered rapidly and efficiently to artificial liposomes from these complexes. In these delivery experiments, the holin exhibits efficient membrane-permeabilizing activity. The S107 antiholin can block formation of the hypersolubilization complexes, suggesting that their formation is related to an oligomerization step intrinsic to holin function.

Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.

KaiA is a two-domain circadian clock protein in cyanobacteria, acting as the positive element in a feedback loop that sustains the oscillation. The structure of the N-terminal domain of KaiA is that of a pseudo -receiver, similar to those of bacterial response regulators, which likely interacts with components of the clock-resetting pathway. The C-terminal domain of KaiA is highly conserved among cyanobacteria and enhances the autokinase activity of KaiC. Here we present the NMR structure of the C-terminal domain of KaiA from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. This domain adopts a novel all α-helical homodimeric structure. Several mutations known to affect the period of the circadian oscillator are shown to be located at an exposed groove near the dimer interface. This NMR structure and a 21-Å-resolution electron microscopy structure of the hexameric KaiC particle allow us to postulate a mode of KaiA–KaiC interaction, in which KaiA binds a linker region connecting two globular KaiC domains.

In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis . Each subunit of the homodimer contains a “motor” domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.

We have studied the binding sites of the electron donor and acceptor proteins of vascular plant photosystem I by electron microscopy/crystallography. Previously, we identified the binding site for the electron acceptor (ferredoxin). In this paper we complete these studies with the characterization of the electron donor (plastocyanin) binding site. After cross-linking, plastocyanin is detected using Fourier difference analysis of two dimensionally ordered arrays of photosystem I located at the periphery of chloroplast grana. Plastocyanin binds in a small cavity on the lumenal surface of photosystem I, close to the center and with a slight bias toward the PsaL subunit of the complex. The recent release of the full coordinates for the cyanobacterial photosystem I reaction center has allowed a detailed comparison between the structures of the eukaryotic and prokaryotic systems. This reveals a very close homology, which is particularly striking for the lumenal side of photosystem I.

Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo-3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm(2). Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex. (Blood. 2002;99:1215-1223)

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked ‘grana’ regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA A receptors are inhibitory. In this study, a fragment of the human glycine receptor α1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli . This fragment is highly homologous in sequence to the corresponding fragment of the GABA A receptor. The recombinant fragment was found to have stable β-rich secondary structure, similar to that found for the homologous GABA A receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA A receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo . Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are approximately 80 x 100 A. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 5' untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).

www.microscopy-today.com • 2010 March mismatch between the immersion oil and the specimen, or local differences in RI within the specimen, cause distortion and a loss of both resolution and fluorescence signal intensity. Pollen grains are a challenging type of specimen because the cellular content of raw pollen is highly autofluorescent and optically inhomogeneous, which leads to light absorption, scattering, and spherical aberration. Typically, only the half of the pollen grain closest to the objective can be imaged at good resolution in single-photon fluorescence microscopy. We have employed sample preparation and mounting techniques that allow single-photon 3D confocal imaging of even relatively large grains (more than 50 mm in diameter) without excessive loss of resolution toward the bottom of the 3D dataset. Two key elements of our technique are pollen extraction and refractive index matching. Introduction The study of pollen is important in paleoecology, paleontology, archeology, and forensics. The outer shell of a pollen grain bears characteristic features that help in determining the species or genus of origin. To view the pollen grain from several angles in a light microscope, the sample is usually mounted in glycerol and rolled on the microscopic slide by pushing the coverslip. For teaching, training, and also for establishing databases of pollen images, it is desirable to obtain three-dimensional datasets that allow rotation and viewing from any angle and reslicing in the computer. Although the scanning electron microscopy provides sufficient depth of field and high resolution of surface features [1], without tilting or rotating the specimen, images from the SEM show only one side of the pollen grain at a time (Figure 1). In contrast, confocal fluorescence microscopy permits optical sectioning and 3D reconstruction of a single pollen grain and allows imaging of not only the surface, but also the internal structure of the pollen grain, albeit at resolution limited by diffraction, that is, typically ~ 0.2 μm in XY and ~ 0.5μm in Z. To achieve this resolution, oil immersion objectives with high numerical aperture must be used. Oil immersion objectives achieve their nominal resolution only when used for specimens with a refractive index (RI) that is homogeneous and equal to that of the immersion oil, that is, η=1.515. An RI 3D Confocal Imaging of Pollen

In many biological membranes, the major lipids are “non-bilayer lipids,” which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that thein vitroassociation of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a/b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.

The type A gamma-aminobutyric acid (GABA(A)) receptor plays a major role in inhibitory synaptic transmission in the central nervous system. A fragment consisting of residues Cys166 to Leu296 of the alpha1 subunit of the GABA(A) receptor was overexpressed in Escherichia coli and was found to have stable beta-rich structures. Here, results from laser scattering, gel electrophoresis and electron microscopy demonstrated that this recombinant protein formed rosette-like homo-oligomers, mainly pentamers in solution. Therefore, the fragment apparently provides a valuable model system for studying the pentameric holoreceptor assembly. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fragment showed that disulfide bonds formed between monomers contributed to the oligomerization of the fragment. The fact that this fragment alone could form pentamers in vitro strongly suggests that amino acid residues located within the Cys166-Leu296 region of the alpha1 subunit may contribute to the oligomerization of GABA(A) receptor in vivo.

Coagulation factor VIII binds to negatively charged platelets prior to assembly with the serine protease, factor IXa, to form the factor X-activating enzyme (FX-ase) complex. The macromolecular organization of membrane-bound factor VIII has been studied by electron crystallography for the first time. For this purpose two-dimensional crystals of human factor VIII were grown onto phosphatidylserine-containing phospholipid monolayers, under near to physiological conditions (pH and salt concentration). Electron crystallographic analysis revealed that the factor VIII molecules were organized as monomers onto the lipid layer, with unit cell dimensions: a = 81.5A, b = 67.2 A, gamma = 66.5 degrees, P1 symmetry. Based on a homology-derived molecular model of the factor VIII (FVIII) A domains, the FVIII projection structure solved at 15-A resolution presents the A1, A2, and A3 domain heterotrimer tilted approximately 65 degrees relative to the membrane plane. The A1 domain is projecting on top of the A3, C1, and C2 domains and with the A2 domain protruding partially between A1 and A3. This organization of factor VIII allows the factor IXa protease and epidermal growth factor-like domain binding sites (localized in the A2 and A3 domains, respectively) to be situated at the appropriate position for the binding of factor IXa. The conformation of the lipid-bound FVIII is therefore very close to that for the activated factor VIIIa predicted in the FX-ase complex.

Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope.

We describe a procedure to obtain structural data of biological macromolecules from small 2-D crystals using cryo-electron crystallography. The procedure has been applied to a membrane-embedded multi-protein complex (photosystem II). This complex, under certain conditions, forms 2-D crystals in its native membrane which are composed of many small mosaic patches and therefore represents an ideal test specimen. By averaging in reciprocal space over more than 50 crystalline patches, it was possible to calculate a projection map using data approaching 1.3 nm resolution while in the Fourier transforms of individual patches only reflections up to the 4th order were readily identifiable. The efficiency of the procedure was evaluated by examining the fidelity of (i) the rotational alignment of patches prior to merging, (ii) the space group assignment, (iii) phase and amplitude extraction, and (iv) the correction of the structure factors for the effects of the contrast transfer function (CTF).

The efficiency of electron crystallographic analysis for 3D structure determination has been demonstrated for an increasing number of proteins and protein complexes. Here, we report cryo-electron crystallography data leading to the first 3D structure of unstained, frozen-hydrated photosystem II (PSII), a multi-protein, membrane-integral complex comprised of more than 20 polypeptides. These data are directly comparable with the 3D structure of negatively stained PSII and reveal a high correlation between the two types of specimens at 3 nm resolution, confirming the earlier assignments of subunits to well-defined domains as well as the functionally important intramolecular cavity. The cavity is at least partially capped on the lumenal surface of the complex and opens out to a 3-nm diameter internal water-filled space that dominates the lumenal side of PSII. The data are discussed in the light of recent studies on PSII subcomplexes. It is concluded that the fidelity of data on transmembrane proteins in negative stain is mainly affected by a flattening of the specimen, partial-depth staining and limited stain penetration into the lipid bilayer.

Photosystem II (PSII) is a protein-pigment complex situated in the thylakoid membranes of plants and cyanobacteria where it catalyses the conversion of light into chemical energy. This energy is used to extract electrons from water, during which process oxygen is evolved. Owing to its extreme fragility and the large number of polypeptides (>20) it is composed of, the complex has so far proven recalcitrant to high-resolution structural studies. Cryo-electron crystallography of 2-D crystals (a = 15.4nm, b = 23.1nm, γ = 97.2°, p1) comprising in situ PSII revealed the first projection structure of the native complex. The unit cell contain one monomeric complex in which three central domains straddle an elongated intramolecular cavity. In conjunction with earlier data, these central domains were assigned to the reaction centre core subunits of PSII consisting of CP43, CP47, the reaction centre heterodimer D1/D2 and cytochrome b-559. The data are discussed in view of the evolution of reaction centres from anoxygenic to oxygenic photosynthesis.