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Nuclear pore complexes (NPCs) span the 2 membranes of the nuclear envelope (NE) and facilitate nucleocytoplasmic exchange of macromolecules. NPCs have a roughly tripartite structural organization with the so-called nuclear basket emanating from the NPC scaffold into the nucleoplasm. The nuclear basket is composed of the 3 nucleoporins Nup153, Nup50, and Tpr, but their specific role for the structural organization of this NPC substructure is, however, not well established. In this study, we have used thin-section transmission electron microscopy to determine the structural consequences of altering the expression of Nup153 in human cells. We show that the assembly and integrity of the nuclear basket is not affected by Nup153 depletion, whereas its integrity is perturbed in cells expressing high concentrations of the zinc-finger domain of Nup153. Moreover, even mild over-expression of Nup153 is coinciding with massive changes in nuclear organization and it is the excess of the zinc-finger domain of Nup153 that is sufficient to induce these rearrangements. Our data indicate a central function of Nup153 in the organization of the nucleus, not only at the periphery, but throughout the entire nuclear interior. [ABSTRACT FROM AUTHOR]

Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape. [ABSTRACT FROM AUTHOR]

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface. [ABSTRACT FROM AUTHOR]

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (αβγδ)4. PhK comprises two arched octameric (αβγδ)2 lobes that are oriented back-to-back with overall D2 symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 Å. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90° instead of 68°, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure. [ABSTRACT FROM AUTHOR]

Phosphorylase kinase (PhK), a 1.3-MDa (αβγδ)4 hexadecameric complex, is a Ca2+-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (αβγδ)2 octameric lobes that are oriented back-to-back with overall D2 symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca2+ by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca2+ in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca2+-activated PhK. Although the overall dimensions of the complex were not affected by Ca2+, the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca2+-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca2+-activated conformers of PhK are consistent with Ca2+-induced conformational changes in both the lobes and the interlobal bridges. [ABSTRACT FROM AUTHOR]

The presence of β-sheets in the core of amyloid fibrils raised questions as to whether or not β-sheet-containing proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the molecular structure of amyloid fibrils differs more generally from the β-sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the ϕ/ψ dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the β-sheet twist. These data imply that amyloid fibril formation from native β-sheet proteins can involve a substantial structural reorganization. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS. [Copyright &y& Elsevier]

A mutant of Synechocystis PCC 6803 lacking the PsaL subunit of photosystem I (PSI) has been grown in iron-deficient media to induce the expression of the isiA gene, which encodes the chlorophyll a-binding protein CP43′. The purpose of this was to establish whether or not the formation of an 18-mer CP43′-PSI supercomplex reported for wild type Synechocystis cells [Nature 412 (2001) 743–745] was dependent on the trimeric conformation of the cyanobacterial PSI reaction centre. Structural characterisation by electron microscopy and single particle image analysis has revealed that the PsaL-mutant does not form trimers of PSI. However, despite this, CP43′ was found to associate with the PSI monomer. The PSI monomer bound six or seven copies of CP43′ along one edge of the PSI monomer and can be compared with one segment of the trimeric 18-mer CP43′-PSI supercomplex. We therefore conclude that the trimeric nature of cyanobacterial PSI is not required for the assembly of the CP43′ antenna system under iron-deficient conditions. [Copyright &y& Elsevier]

Swm1p, a subunit of the APC cyclosome, was originally identified for its role in the later stages of the sporulation process and is required for spore wall assembly. In addition, this protein is required to maintain cell wall integrity in vegetative cells during growth at high temperature. Electron microscopy analyses of mutant cells grown at the restrictive temperature in the absence of osmotic support show that the cell wall is clearly abnormal, with large number of discontinuities that may be responsible for the observed lysis. The mutant cells show a 7-fold reduction in glucan synthase activity during growth at 38 °C and a 3.5-fold increase in the chitin content of the cell wall. The chitin is deposited in a delocalized manner all over the cell wall, where it accumulates in patches in abnormal regions. The excess chitin is mainly synthesized by the action of chitin synthase III (Chs3p), since it disappears in the swm1 chs3 double-mutant. [Copyright &y& Elsevier]

A large barrier in the way to obtaining high-resolution structures of eukaryotic ion channels remains the expression and purification of sufficient amounts of channel protein to carry out crystallization trials. In the absence of crystals, the main available source of structural information has been electron microscopy (EM), which is well suited to the visualization of isolated macromolecular complexes and their conformational changes. The recently published EM structures outline native conformations of eukaryotic cation channels that until now have eluded crystallization. According to these results, homo-tetrameric K+ channels have a distinct two-layer architecture with connectors conjoining the two layers, while the pseudo-tetrameric Ca2+ or Na+ channels are more globular and have flexible surface loops, which makes the identification of subunits complicated. Subunits can be identified using atomic structure docking into the EM maps, labeling, or deletion studies. [Copyright &y& Elsevier]

The N-terminal domain of HIV-1 glycoprotein 41,000 (gp41) participates in viral fusion processes. Here, we use physical and computational methodologies to examine the secondary structure of a peptide based on the N terminus (FP; residues 1-23) in aqueous and detergent environments. 12C-Fourier transform infrared (FTIR) spectroscopy indicated greater α-helix for FP in lipid-detergent sodium dodecyl sulfate (SDS) and aqueous phosphate-buffered saline (PBS) than in only PBS. 12C-FTIR spectra also showed disordered FP conformations in these two environments, along with substantial β-structure for FP alone in PBS. In experiments that map conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using FP peptides labeled with 13C-carbonyl. 13C-FTIR results on FP in SDS at low peptide loading indicated α-helix (residues 5 to 16) and disordered conformations (residues 1-4). Because earlier 13C-FTIR analysis of FP in lipid bilayers demonstrated α-helix for residues 1-16 at low peptide loading, the FP structure in SDS micelles only approximates that found for FP with membranes. Molecular dynamics simulations of FP in an explicit SDS micelle indicate that the fraying of the first three to four residues may be due to the FP helix moving to one end of the micelle. In PBS alone, however, electron microscopy of FP showed large fibrils, while 13C-FTIR spectra demonstrated antiparallel β-sheet for FP (residues 1-12), analogous to that reported for amyloid peptides. Because FP and amyloid peptides each exhibit plaque formation, α-helix to β-sheet interconversion, and membrane fusion activity, amyloid and N-terminal gp41 peptides may belong to the same superfamily of proteins. [ABSTRACT FROM AUTHOR]

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The β-strand-rich single chain Fv fragment (scFv) and α-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is α-helix rich, are predominated by α-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the α-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins. [Copyright &y& Elsevier]

Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore-forming protein family formed by the single-component α-hemolysin and the bicomponent leukocidins and γ-hemolysins. The last comprise two protein elements, S and F, that co-operatively form the active toxin. α-Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more specifically involved in a few diseases. X-ray crystallography of the α-hemolysin pore has shown it is a mushroom-shaped, hollow heptamer, almost entirely consisting of β-structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre-pore can trigger Ca2+-mediated activation of some white cells, initiating an inflammatory response. Within the bicomponent toxins, γ-hemolysins define three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like α-hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. γ-Hemolysin pores seem to be organized as α-hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry. [Copyright &y& Elsevier]

Cyanobacteria, unlike plants and green algae, do not contain chlorophyll (Chl) b. This is because of the absence of the cao gene which encodes the enzyme that catalyses a two step oxygenation of chlorophyllide a to chlorophyllide b. Recently, however, the cao gene of higher plants was engineered into Synechocystis PCC 6803 leading to Chl b synthesis in this cyanobacterium [Satoh et al., J. Biol. Chem. 276 (2001) 4293–4297]. Here we use this same cao-plus mutant to show that Chl b can bind to the CP43′ protein, expressed in cells exposed to low iron levels, which normally binds Chl a only. In so doing CP43′ is changed to a Chl a/Chl b-binding protein and in this respect resembles the closely related Chl a/Chl b-binding Pcb protein of prochlorophytes (green oxyphotobacteria). The results emphasise the possibility of using an in vitro system to elucidate factors which control the binding of these two different forms of chlorophylls to the six transmembrane helical light-harvesting proteins of oxygenic photosynthetic organisms. [Copyright &y& Elsevier]

Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) from Pyrobaculum aerophilum ( Pae) and Methanobacterium thermautotrophicum ( Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide-bonded 14-mers or sub-heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head-to-tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14-mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 Å-resolution structure of Mth SmAP1 bound to uridine-5′-monophosphate (UMP) reveals conserved ligand-binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus ( Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel-shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single-stranded nucleic acid-binding activity. [ABSTRACT FROM AUTHOR]

Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially α-helical but converts rapidly to a β-sheet structure and large quantities of fibrils form. In SDS above the critical micellar concentration the peptide adopts a stable α-helical structure and no fibrils are observed. These findings demonstrate the sensitivity of fibril formation to solution conditions and suggest a possible role for membrane components in amyloid fibril formation in living systems. [Copyright &y& Elsevier]

β2-Microglobulin (β2-m) is a major component of dialysis-related amyloid fibrils. Although recombinant β2-m forms needle-like fibrils by in vitro extension reaction at pH 2.5, reduced β2-m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of β2-m in the acid-denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. {1H}-15N NOE at the regions between the two cysteine residues (Cys25-Cys80) revealed a marked difference in the pico- and nanosecond time scale dynamics between that the acid-denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, ΔCα, ΔCO, and ΔHα, and 3JHNHα coupling constants indicated that both the oxidized and reduced β2-m at pH 2.5 have marginal α-helical propensity at regions close to the C-terminal cysteine, although it is a β-sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of β2-m, and that the marginal helical propensity at the C-terminal regions might play a role in modifying this potential. [ABSTRACT FROM AUTHOR]

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of photosystem I and light-harvesting complex I from the unicellular green alga Chlamydomonas reinhardtii. The complex is a monomer, has longest dimensions of 21.3 and 18.2 nm in projection, and is significantly larger than the corresponding complex in spinach. Comparison with photosystem I complexes from other organisms suggests that the complex contains about 14 light-harvesting proteins, two or three of which bind at the side of the PSI-H subunit. We suggest that special light-harvesting I proteins play a role in the binding of phosphorylated light-harvesting complex II in state 2. [Copyright &y& Elsevier]

Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the α-helical state that is formed upon binding to a hydrophobic solid, and the β-sheet state, which is formed at the air-water interface. Here, we show that the formation of the β-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the α-helical state of SC3. The second intermediate is rich in β-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets. [ABSTRACT FROM AUTHOR]

Aggregation of expanded polyglutamine (polyQ) seems to be the cause of various genetic neurodegenerative diseases. Relatively little is known as yet about the polyQ structure and the mechanism that induces aggregation. We have characterised the solution structure of polyQ in a proteic context using a model system based on glutathione S-transferase fusion proteins. A wide range of biophysical techniques was applied. For the first time, nuclear magnetic resonance was used to observe directly and selectively the conformation of polyQ in the pathological range. We demonstrate that, in solution, polyQs are in a random coil conformation. However, under destabilising conditions, their aggregation behaviour is determined by the polyQ length. [Copyright &y& Elsevier]

Amide hydrogen exchange and mass spectrometry have been used to study the pH-induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D2O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi-threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo-electron microscopy and image reconstruction. Structural changes in the extended N- and C-terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids. [ABSTRACT FROM AUTHOR]