Connexin 43 maintains tissue polarity and regulates mitotic spindle orientation in the breast epithelium.

Cell-cell communication is essential for tissue homeostasis, but its contribution to disease prevention remains to be understood. We demonstrate the involvement of connexin 43 (Cx43, also known as GJA1) and related gap junction in epithelial homeostasis, illustrated by polarity-mediated cell cycle entry and mitotic spindle orientation (MSO). Cx43 localization is restricted to the apicolateral membrane of phenotypically normal breast luminal epithelial cells in 3D culture and in vivo Chemically induced blockade of gap junction intercellular communication (GJIC), as well as the absence of Cx43, disrupt the apicolateral distribution of polarity determinant tight junction marker ZO-1 (also known as TJP1) and lead to random MSO and cell multilayering. Induced expression of Cx43 in cells that normally lack this protein reestablishes polarity and proper MSO in 3D culture. Cx43-directed MSO implicates PI3K-aPKC signaling, and Cx43 co-precipitates with signaling node proteins ß-catenin (CTNNB1) and ZO-2 (also known as TJP2) in the polarized epithelium. The distribution of Cx43 is altered by pro-inflammatory breast cancer risk factors such as leptin and high-fat diet, as shown in cell culture and on tissue biopsy sections. The control of polarity-mediated quiescence and MSO may contribute to the tumor-suppressive role of Cx43.

Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB.

Spinae are tubular surface appendages broadly found in Gram-negative bacteria. Little is known about their architecture, function or origin. Here, we report structural characterization of the spinae from marine bacteria Roseobacter sp. YSCB. Electron cryo-tomography revealed that a single filament winds into a hollow flared base with progressive change to a cylinder. Proteinase K unwound the spinae into proteolysis-resistant filaments. Thermal treatment ripped the spinae into ribbons that were melted with prolonged heating. Circular dichroism spectroscopy revealed a dominant beta-structure of the spinae. Differential scanning calorimetry analyses showed three endothermic transformations at 50-85°C, 98°C and 123°C, respectively. The heating almost completely disintegrated the spinae, abolished the 98°C transition and destroyed the beta-structure. Infrared spectroscopy identified the amide I spectrum maximum at a position similar to that of amyloid fibrils. Therefore, the spinae distinguish from other bacterial appendages, e.g. flagella and stalks, in both the structure and mechanism of assembly.

Draft genome sequence of the purple photosynthetic bacterium Phaeospirillum molischianum DSM120, a particularly versatile bacterium.

Here we present the draft genome sequence of the versatile and adaptable purple photosynthetic bacterium Phaeospirillum molischianum DSM120. This study advances the understanding of the adaptability of this bacterium, as well as the differences between the Phaeospirillum and Rhodospirillum genera.

Automated quantification and reconstruction of collagen matrix from 3D confocal datasets.

The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered-light confocal images of collagen gels. The computed quantitative data allowed us to create surface-rendered 3D images of the investigated sample.

The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB.

The Tol-Pal system of Escherichia coli is required for the maintenance of outer membrane stability. Recently, proton motive force (pmf) has been found to be necessary for the co-precipitation of the outer membrane lipoprotein Pal with the inner membrane TolA protein, indicating that the Tol-Pal system forms a transmembrane link in which TolA is energized. In this study, we show that both TolQ and TolR proteins are essential for the TolA-Pal interaction. A point mutation within the third transmembrane (TM) segment of TolQ was found to affect the TolA-Pal interaction strongly, whereas suppressor mutations within the TM segment of TolR restored this interaction. Modifying the Asp residue within the TM region of TolR indicated that an acidic residue was important for the pmf-dependent interaction of TolA with Pal and outer membrane stabilization. Analysis of sequence alignments of TolQ and TolR homologues from numerous Gram-negative bacterial genomes, together with analyses of the different tolQ-tolR mutants, revealed that the TM domains of TolQ and TolR present structural and functional homologies not only to ExbB and ExbD of the TonB system but also with MotA and MotB of the flagellar motor. The function of these three systems, as ion potential-driven molecular motors, is discussed

Adsorption of avidin on microfabricated surfaces for protein biochip applications.

The adsorption of the protein avidin from hen egg white on patterns of silicon dioxide and platinum surfaces on a microchip and the use of fluorescent microscopy to detect binding of biotin are described. A silicon dioxide microchip was formed using plasma-enhanced chemical vapor deposition while platinum was deposited using radiofrequency sputtering. After cleaning using a plasma arc, the chips were placed into solutions containing avidin or bovine serum albumin. The avidin was adsorbed onto the microchips from phosphate-buffered saline (PBS) or from PBS to which ammonium sulfate had been added. Avidin was also adsorbed onto bovine serum albumin (BSA)-coated surfaces of oxide and platinum. Fluorescence microscopy was used to confirm adsorption of labeled protein, or the binding of fluorescently labeled biotin onto previously adsorbed, unlabeled avidin. When labeled biotin in PBS was presented to avidin adsorbed onto a BSA-coated microchip, the fluorescence signal was significantly higher than for avidin adsorbed onto the biochip alone. The results show that a simple, low-cost adsorption process can deposit active protein onto a chip in an approach that has potential application in the development of protein biochips for the detection of biological species.

Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme.

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K(i) approximately 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.

Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay.

Pheromone-binding proteins (PBPs), located in the sensillum lymph of pheromone-responsive antennal hairs, are thought to transport the hydrophobic pheromones to the chemosensory membranes of olfactory neurons. It is currently unclear what role PBPs may play in the recognition and discrimination of species-specific pheromones. We have investigated the binding properties and specificity of PBPs from Mamestra brassicae (MbraPBP1), Antheraea polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal cavity to mimic that of BmorPBP, using the fluorescence probe 1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however, no binding was observed with either BmorPBP or Mbra1-M6. The latter result indicates that relatively limited modifications to the PBP cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA from the MbraPBP1- and ApolPBP1-binding sites, without, however, any evidence of specificity for their physiologically relevant pheromones. Moreover, some fatty acids are also able to compete with AMA binding. These findings bring into doubt the currently held belief that all PBPs are specifically tuned to distinct pheromonal compounds.

Organisation and evolution of the tol-pal gene cluster.

The genomic context and phylogenetic distribution of the tol-pal gene cluster and homologues to its various components have been investigated. The structure of this operon is well conserved across the gram negative bacteria, and the machine encoded by these genes probably evolved with the appearance of gram negative bacteria. Since the evolutionary appearance of the operon some species appear to have lost the genes. These bacteria seem to fall into two classes, namely obligate intracellular parasites and bacteria that produce large numbers of outer membrane vesicles. The evolution of the alphabeta and gamma proteobacteria was accompanied by the association of an additional gene (ybgC) with the operon. Several coincidences of genomic context argue for an important role of the tol-pal operon in cell envelope maintenance. Genes homologous to tolQ and tolR proved to be very widespread being found throughout the eubacteria, and one example in the archea, this distribution argues for an ancient origin of these genes. The genomic context of these genes often suggests a role in micronutrient uptake. Interestingly in all the cases examined the tolQ and tolR genes or their homologues appear to be present as a pair, with a potential for a tight translational regulation.

Proton motive force drives the interaction of the inner membrane TolA and outer membrane pal proteins in Escherichia coli.

The Tol-Pal system of the Escherichia coli envelope is formed from the inner membrane TolQ, TolR and TolA proteins, the periplasmic TolB protein and the outer membrane Pal lipoprotein. Any defect in the Tol-Pal proteins or in the major lipoprotein (Lpp) results in the loss of outer membrane integrity giving hypersensitivity to drugs and detergents, periplasmic leakage and outer membrane vesicle formation. We found that multicopy plasmid overproduction of TolA was able to complement the membrane defects of an lpp strain but not those of a pal strain. This result indicated that overproduced TolA has an envelope-stabilizing effect when Pal is present. We demonstrate that Pal and TolA formed a complex using in vivo cross-linking and immunoprecipitation experiments. These results, together with in vitro experiments with purified Pal and TolA derivatives, allowed us to show that Pal interacts with the TolA C-terminal domain. We also demonstrate using protonophore, K+ carrier valinomycin, nigericin, arsenate and fermentative conditions that the proton motive force was coupled to this interaction.

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels.

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.

Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro.

The development of the next generation of biomaterials for restoration of tissues and organs (i.e., tissue engineering) requires a better understanding of the extracellular matrix (ECM) and its interaction with cells. Extracellular matrix is a macromolecular assembly of natural biopolymers including collagens, glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins. Interestingly, several ECM components have the ability to form three-dimensional (3D), supramolecular matrices (scaffolds) in vitro by a process of self-directed polymerization, "self-assembly". It has been shown previously that 3D matrices with distinct architectural and biological properties can be formed from either purified type I collagen or a complex mixture of interstitial ECM components derived from intestinal submucosa. Unfortunately, many of the imaging and analysis techniques available to study these matrices either are unable to provide insight into 3D preparations or demand efforts that are often prohibitory to observations of living, dynamic systems. This is the first report on the use of reflection imaging at rapid time intervals combined with laser-scanning confocal microscopy for analysis of structural properties and kinetics of collagen and ECM assembly in 3D. We compared time-lapse confocal reflection microscopy (TL-CRM) with a well-established spectrophotometric method for determining the self-assembly properties of both purified type I collagen and soluble interstitial ECM. While both TL-CRM and spectrophotometric techniques provided insight into the kinetics of the polymerization process, only TL-CRM allowed qualitative and quantitative evaluation of the structural parameters (e.g., fibril diameter) and 3D organization (e.g., fibril density) of component fibrils over time. Matrices formed from the complex mixture of soluble interstitial ECM components showed an increased rate of assembly, decreased opacity, decreased fibril diameter, and increased fibril density compared to that of purified type I collagen. These results suggested that the PG/GAG components of soluble interstitial ECM were affecting the polymerization of the component collagens. Therefore, the effects of purified and complex mixtures of PG/GAG components on the assembly properties of type I collagen and interstitial ECM were evaluated. The data confirmed that the presence of PG/GAG components altered the kinetics and the 3D fibril morphology of assembled matrices. In summary, TL-CRM was demonstrated to be a new and useful technique for analysis of the 3D assembly properties of collagen and other natural biopolymers which requires no specimen fixation and/or staining.

Exchanging cofactors in the core antennae from purple bacteria: structure and properties of Zn-bacteriopheophytin-containing LH1.

The core light-harvesting LH1 complex of Rhodospirillum rubrum consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll (BChl) a molecule. In this work, we describe a technique that allows the replacement of the natural, Mg BChl a cofactors present in this protein by Zn-bacteriopheophytin (Zn-Bpheo). This technique makes use of the well-characterized, reversible dissociation of LH1 induced by the detergent beta-octylglucoside. Incubation of partially dissociated LH1 with exogeneous pigments induces an equilibrium between the protein-bound BChl and the exogeneous pigment. This results in the binding of chemically modified pigments to LH1, in amounts which depend on the pigment composition and concentration of the exchange buffer. This method can yield information on the relative affinities of the LH1 protein-binding sites for the different pigments BChl and Zn-Bpheo and can also be used to obtain fully reassociated LH1 proteins, with a variable content of modified pigment, which may be precisely monitored. Absorption and FT-Raman spectroscopy indicate that this exchange procedure leads to LH1 proteins containing modified pigments, but retaining a binding site structure identical to that of native LH1. Furthermore, examination of the binding curves suggests that there are two distinguishable binding sites, probably corresponding to the two polypeptides, with very different properties. One of these two binding sites shows a marked preference for Zn-Bpheo over BChl, while the other binding site appears to prefer BChl.

Detergents modulate dimerization, but not helicity, of the glycophorin A transmembrane domain.

Understanding how the lipid environment influences transmembrane helix association requires thermodynamic measurements that can be interpreted in terms of specific chemical interactions. We have used Förster resonance energy transfer to measure dimerization of the glycophorin A transmembrane helix in detergent micelles. The observed Kd is at least two orders of magnitude weaker in sodium dodecyl sulfate than it is in zwitterionic detergents. In contrast, neither dimerization nor the detergent affects the secondary structure of the glycophorin A helix as measured by far-UV circular dichroism. These measurements support a long standing assumption about the glycophorin A transmembrane domain, that detergents uncouple helix formation from helix dimerization. The approach is applicable to a variety of systems in diverse environments, extending our ability to measure how interactions with complex solvents affect the thermodynamics of oligomerization.

Conformation of bacteriochlorophyll molecules in photosynthetic proteins from purple bacteria.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.

The effect of pressure on the bacteriochlorophyll a binding sites of the core antenna complex from Rhodospirillum rubrum.

In this paper we examine the effect of pressure on the absorption spectrum and binding site of the core antenna complex from the photosynthetic bacterium Rhodospirillum rubrum. Absorption spectra and Raman spectra in preresonance with the Qy transition of the bacteriochlorophyll a were studied at pressures up to 625 MPa. In agreement with previous work we observe a pressure-induced red shift and broadening of the absorption spectrum. We show that at these pressures the pigments within the protein matrix at room temperature experience little if any distortion, and the hydrogen-bonding network involving the C2 and C9 carbonyl groups of the pigment molecules are undisturbed. Having shown the lack of sensitivity to pressure of the binding site interactions, which are known to modulate the absorption spectrum, we feel that it is relatively safe to attribute the pressure-induced red shift broadly to solvatochromic effects and, in particular, to the modulation of the pigment-pigment interactions by the pressure. This paper represents the first vibrational study of photosynthetic complexes at high pressure and the first application of FT Raman spectroscopy to biological molecules at high pressure.

Hydrogen bonding and circular dichroism of bacteriochlorophylls in the Rhodobacter capsulatus light-harvesting 2 complex altered by combinatorial mutagenesis.

We have investigated the spectroscopic properties of two classes of light-harvesting 2 (LH2, B800-850) mutants of Rhodobacter capsulatus obtained by combinatorial mutagenesis to the C-terminal half of the beta-apoprotein: a pseudoLH2 (pLH2) class, in which the 800-nm absorption was normal but the 850-nm peak was blue-shifted by up to 14 nm, and the other a pseudoLH1 (pLH1) class, which lacked the 800-nm absorption band and showed 850-nm absorption red-shifts of up to 30 nm. In several of the pLH1 antennae, carotenoid depletion contributed to the phenotype, while in the pLH2 complexes there was some carotenoid enrichment. A number of mutants from each class have also been characterized by low-temperature absorption and fluorescence spectroscopy, resonance Raman spectroscopy, and circular dichroism. In all of the mutants investigated, the B850 bacteriochlorophyll a binding site remained intact, conserving both the hydrogen bonding environment of the chromophores and their conformation and liganding. In contrast, the intensity of the CD spectra of pLH1 complexes was considerably reduced, relative to that of wild-type or pLH2 complexes, consistent with alterations in the interactions between pigments and in their relative orientation. Elevated fluorescence polarization over the red wing of the B850 band in the pLH2 complexes indicated a reduction of exciton mobility within the ring of BChl molecules. Possible structural alterations governing the spectral properties of the different mutants are discussed.

Transmembrane helix stability: the effect of helix-helix interactions studied by Fourier transform infrared spectroscopy.

We have measured, using infrared spectroscopy, the hydrogen/deuterium exchange rates of the amide protons in the photosynthetic antenna of Rhodospirillum rubrum. These measurements were made not only on the intact protein in detergent solution but also on two dissociated forms (B820 and B777). We have, on the basis of our knowledge of the structure of this protein, been able to assign the various groups of amide protons that exchange with different time constants to distinct regions of the protein. The most protected group of protons that we observe exchanging with time constants near 6000 min we assign to the transmembrane helices. The slow exchange rates measured for the amide protons of the transmembrane helices of this protein in detergent solution may indicate a destabilization of the helices in detergent solution compared with the membrane. This group of protons is progressively destabilized by stepwise dissociation of the antenna protein, and this destabilization is greater than we can account for by increases in solvent accessibility. We suggest that the observed loss of amide proton protection in the transmembrane helices as they are dissociated might be due to an increase in the helix flexibility and breathing motions as interactions between helices are reduced.

Heterologous expression of genes encoding bacterial light-harvesting complex II in Rhodobacter capsulatus and Rhodovulum sulfidophilum.

In the present work we report the high-level expression of foreign genes encoding the light-harvesting (LHII) membrane-spanning polypeptides in photosynthetic bacteria. To do this we first constructed three deletion strains of Rhodovulum (Rhv.) sulfidophilum in which all or part of the puc operon, encoding the peripheral light-harvesting proteins, is missing. To investigate the heterologous expression of the light-harvesting polypeptides from Rb. capsulatus in Rhv. sulfidophilum and vice versa we have reintroduced functional foreign LH genes into these and equivalent strains of Rhodobacter (Rb.) capsulatus. In some cases very high levels of expression were obtained (85%) of those observed in the wild type), while in other cases much lower expression was observed; possible reasons for these differences are discussed. The heterologously expressed proteins were shown to contain normal pigment-binding sites and to be normally and functionally integrated within the host photosynthetic apparatus. The results indicate that heterologous proteins are able to assemble properly and enter into the same protein-protein interactions as their analogs originally present in the host strain.