RESUMEN
Cyclic [3]spirobifluorenylene with bulky alkyl groups at the ends (1) was designed and synthesized to investigate the electron transfer phenomena in a π-conjugated system including orthogonal π-conjugated chains. The three bifluorenyl units in 1 are conjugated to each other via spiro-conjugation, resulting in the splitting of the HOMO levels to a small extent. Therefore, the SOMO-HOMO gap of the radical cation species is small, which is considered to allow the facile intramolecular electron transfer. The electronic properties of 1 and its partial structures were characterized by absorption and fluorescence measurements and electrochemical analysis. From the electrochemical oxidation, the interchain Coulombic repulsion was observed. In the TD-DFT calculations for the radical cation species of 1, the geometry-featured interchain electronic transitions were visualized by NTO calculations. The radical cation species of 1 generated by chemical oxidation with SbCl5 exhibited a broadened and lower-energy NIR absorption band exceeding 2000 nm. Considering the results of the TD-DFT calculations, the NIR band of the radical cation of 1 was attributed to the intramolecular electron transfer processes among the bifluorenyl units in the macrocycle. ESR experiments also indicated the delocalization of a spin of 1â + in the whole molecule via hole hopping in the ESR time scale at room temperature. This work demonstrates the usefulness of spiro-conjugation as a bridging unit in molecular wires to facilitate smooth electron transfer.
RESUMEN
We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.
Asunto(s)
Proteínas Bacterianas , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante) , Lactobacillaceae , Transducción de Señal , Receptor Toll-Like 2 , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Inmunoglobulina A/inmunología , Interleucina-6/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/aislamiento & purificación , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/farmacología , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/aislamiento & purificación , Adyuvantes Inmunológicos/farmacología , Animales , Ratones , Lactobacillaceae/clasificación , Lactobacillaceae/enzimología , Lactobacillaceae/genética , Lactobacillaceae/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , FN-kappa B/inmunología , Activación Transcripcional/efectos de los fármacosRESUMEN
Recent studies have suggested that in certain environments, tree stems emit methane (CH4 ). This study explored the mechanism of CH4 emission from the stem surfaces of Alnus japonica in a riparian wetland. Stem CH4 emission rates and sap flux were monitored year-round, and fine-root anatomy was investigated. CH4 emission rates were estimated using a closed-chamber method. Sap flux was measured using Granier-type thermal dissipation probes. Root anatomy was studied using both optical and cryo-scanning electron microscopy. CH4 emissions during the leafy season exhibited a diurnally changing component superimposed upon an underlying continuum in which the diurnal variation was in phase with sap flux. We propose a model in which stem CH4 emission involves at least two processes: a sap flux-dependent component responsible for the diurnal changes, and a sap flux-independent component responsible for the background continuum. The contribution ratios of the two processes are season-dependent. The background continuum possibly resulted from the diffusive transport of gaseous CH4 from the roots to the upper trunk. Root anatomy analysis indicated that the intercellular space of the cortex and empty xylem cells in fine roots could serve as a passageway for transport of gaseous CH4 .
Asunto(s)
Alnus , Metano , Metano/análisis , Estaciones del Año , Suelo , Árboles , HumedalesRESUMEN
Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis toward host immune cells. Bifidobacterium infantis MVs consisted of a cytoplasmic membrane, and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.
Asunto(s)
Bifidobacterium longum subspecies infantis , Interleucina-6 , Interleucina-6/metabolismo , Bifidobacterium/metabolismo , Heces/microbiología , Inmunoglobulina ARESUMEN
Dibenzo[g,p]chrysene (DBC), which consists of a twisted naphthalene core with four fused benzene rings, is a promising framework for organic electronic materials. Therefore, the research for structure-property relationships is important for the design of DBC-based materials. Here, the electrochemical and spectroscopic properties of DBC derivatives were investigated, and the effects of substituents and torsion of the naphthalene moiety were examined based on density functional theory (DFT) calculations. All the substituted DBC derivatives showed higher oxidation potentials than that for DBC-H, even for compounds that contained an electron-donating group such as DBC-Me and DBC-SMe. DFT calculations clearly indicate that these higher oxidation potentials are due to the ineffective conjugation of the MeO group, which is oriented perpendicular to the benzene ring because of the steric repulsion of substituents on both sides. More specifically, the inductive effect of the MeO group is dominant rather than the mesomeric effect when the substituent is located at both sides of the MeO group. Concerning the torsion of the naphthalene moiety, the twisting results in a slight increase in the HOMO and a slight lowering of the LUMO. The twisting effect is much smaller than the conjugation effect of the MeO group. Absorption spectra of all the substituted DBC derivatives also showed a red-shift as compared to that for DBC-H. Concerning the luminescence, a strong photoluminescence was observed for DBC-H and DBC-Si.
RESUMEN
Initial attachment and subsequent colonization of the intestinal epithelium comprise critical events allowing enteric pathogens to survive and express their pathogenesis. In enterotoxigenic Escherichia coli (ETEC), these are mediated by a long proteinaceous fiber termed type IVb pilus (T4bP). We have reported that the colonization factor antigen/III (CFA/III), an operon-encoded T4bP of ETEC, possesses a minor pilin, CofB, that carries an H-type lectin domain at its tip. Although CofB is critical for pilus assembly by forming a trimeric initiator complex, its importance for bacterial attachment remains undefined. Here, we show that T4bP is not sufficient for bacterial attachment, which also requires a secreted protein CofJ, encoded within the same CFA/III operon. The crystal structure of CofB complexed with a peptide encompassing the binding region of CofJ showed that CofJ interacts with CofB by anchoring its flexible N-terminal extension to be embedded deeply into the expected carbohydrate recognition site of the CofB H-type lectin domain. By combining this structure and physicochemical data in solution, we built a plausible model of the CofJ-CFA/III pilus complex, which suggested that CofJ acts as a molecular bridge by binding both T4bP and the host cell membrane. The Fab fragments of a polyclonal antibody against CofJ significantly inhibited bacterial attachment by preventing the adherence of secreted CofJ proteins. These findings signify the interplay between T4bP and a secreted protein for attaching to and colonizing the host cell surface, potentially constituting a therapeutic target against ETEC infection.
Asunto(s)
Adhesión Bacteriana , Escherichia coli Enterotoxigénica/química , Proteínas de Escherichia coli/química , Fimbrias Bacterianas/química , Cristalografía por Rayos X , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Escherichia coli Enterotoxigénica/patogenicidad , Escherichia coli K12/química , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Operón , Dominios ProteicosRESUMEN
A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Familia de Multigenes/genética , Shewanella/genética , Proteínas Bacterianas/genética , Membrana Celular/genética , Vesículas Extracelulares/genética , Transporte de Proteínas , Shewanella/metabolismoRESUMEN
Selenite reduction is a key step in the biogeochemical cycle of selenium-an essential trace element for life. A variety of bacteria can transform selenite into elemental selenium nanoparticles on the cell surface via anaerobic respiration or detoxification processes. However, the proteins associated with the uptake of selenite for these processes are poorly understood. In this study, we investigated the role of an outer membrane porin-like protein, ExtI, in selenite permeation in Geobacter sulfurreducens. We demonstrated that selenite uptake and selenium nanoparticle formation were impaired in an extI-deficient strain. A putative rhodanese-like lipoprotein is encoded by an extH gene located immediately upstream of extI in the genome. We showed that ExtH is translocated into inner and outer membranes and that extI deficiency exclusively affects the localization of ExtH in the outer membrane. Coelution of ExtI and ExtH during gel filtration analysis of the outer membrane fraction of wild-type cells suggests a direct protein-protein interaction between them. Taken together, these results lead us to propose a physiological role for ExtI as a selenite channel associated with ExtH in the outer membrane.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Geobacter/metabolismo , Lipoproteínas/metabolismo , Porinas/metabolismo , Ácido Selenioso/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Membrana Celular/metabolismo , Nanopartículas/química , Nanopartículas/ultraestructura , Fracciones SubcelularesRESUMEN
An amphiphilic polypeptide, poly(sarcosine)- b-(l-Leu-Aib)8 (SL16), was reported to self-assemble into vesicles. A GxxxG motif, which is known to induce helix dimerization, is incorporated into the hydrophobic helical block of SL16 to synthesize poly(sarcosine)- b-(l-Leu-Aib)2-(Gly-Aib-l-Leu-Aib-Gly-Aib)-(l-Leu-Aib)3 (SG16). SG16 shows helix association in ethanol at a high concentration and low temperatures, which is not observed with SL16. SG16 self-assembles into vesicles, but are found to be more susceptible to rupture by the addition of Triton X-100 than SL16 vesicles. A mixture of SL16 and SG16 self-assembles into small sheets and micelles likely because of mismatch of the modes of helix association arising from sterical accommodation of iso-butyl groups at the helix-helix interface.
RESUMEN
A novel cyclic hexapeptide composed of l-α-naphthylalanine, d-α-anthrylalanine, and four ß-alanines (CP6) is synthesized and its molecular assembly into peptide nanotubes (PNTs) and the electronic properties arising from one-dimensional arrays of aromatic groups along the PNTs were investigated. CP6 with a combination of l- and d-α-amino acids is designed to self-assemble into PNTs with them stacking on top of each other under the constraint of maximizing the number of intermolecular hydrogen bonds between the cyclic peptides. Upon PNT formation, the respective side chains of l- and d-α-amino acids are aligned in line along the PNTs. The topological arrangement of the anthryl groups being in close proximity in the CP6 PNT is supported by higher photo-excited energy transfer, appearance of the induced Cotton effects, and the promoted photo-dimerization reaction upon PNT formation. AFM observations reveal that PNT bundles with diameters 5-15 nm are dielectric microcrystals having a piezoelectric coefficient of 2-6 pC N-1. Kelvin force microscopy observations show the generation of surface potentials over 100 mV owing to the one-dimensional array of the anthryl groups along PNTs. Incorporation of α-amino acids with opposite chirality into cyclic ß-peptides is therefore an effective molecular design for the nano-architecture of PNTs displaying one-dimensional arrays of chromophores along PNTs.
Asunto(s)
Aminoácidos/química , Nanotubos de Péptidos/química , Péptidos Cíclicos/química , Microscopía de Fuerza Atómica , Naftalenos/químicaRESUMEN
Many insects and Dipterans in particular are known to spend considerable time grooming, but whether these behaviors actually are able to remove pathogenic fungal conidia is less clear. In this study, we examined whether grooming serves to protect flies by reducing the risk of fungal infection in Drosophila melanogaster. First, we confirmed that fungi were removed by grooming. Entomopathogenic, opportunistic, and plant pathogenic fungi were applied on the body surface of the flies. To estimate grooming efficiency, the number of removal conidia through grooming was quantified and we successfully demonstrated that flies remove fungal conidia from their body surfaces via grooming behavior. Second, the roles of gustatory and olfactory signals in fungus removal were examined. The wildtype fly Canton-S, the taste deficiency mutant poxn 70, and the olfactory deficiency mutant orco1 were used in the tests. Comparisons between Canton-S and poxn 70 flies indicated that gustatory signals do not have a significant role in fungal removal via grooming behavior in D. melanogaster. In contrast, the efficiency of conidia removal in orco1 flies was drastically decreased. Consequently, this study indicated that flies rely on mechanical stimulus for the induction of grooming and olfaction for more detailed removal.
Asunto(s)
Drosophila melanogaster/microbiología , Drosophila melanogaster/fisiología , Aseo Animal/fisiología , Hongos Mitospóricos , Olfato/fisiología , Animales , Señales (Psicología) , Interacciones Huésped-Parásitos/fisiologíaRESUMEN
The pectin in plant cell walls consists of three domains: homogalacturonan, rhamnogalacturonan (RG)-I, and RG-II. It is predicted that around 50 different glycosyltransferases are required for their biosynthesis. Among these, the activities of only a few glycosyltransferases have been detected because pectic oligosaccharides are not readily available for use as substrates. In this study, fluorogenic pyridylaminated RG-I-backbone oligosaccharides (PA-RGs) with 3-14 degrees of polymerization (DP) were prepared. Using these oligosaccharides, the activity of RG-I:rhamnosyltransferase (RRT), involved in the biosynthesis of the RG-I backbone diglycosyl repeating units (-4GalUAα1-2Rhaα1-), was detected from the microsomes of azuki bean epicotyls. RRT was found to prefer longer acceptor substrates, PA-RGs with a DP > 7, and it does not require any metal ions for its activity. RRT is located in the Golgi and endoplasmic reticulum. The activity of RRT coincided with epicotyl growth, suggesting that RG-I biosynthesis is involved in plant growth.
Asunto(s)
Pared Celular/metabolismo , Glicosiltransferasas/metabolismo , Pectinas/biosíntesis , Proteínas de Plantas/metabolismo , Biocatálisis , Pared Celular/enzimología , Cromatografía Líquida de Alta Presión , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Espectroscopía de Resonancia Magnética , Oligosacáridos/metabolismo , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Vigna/enzimología , Vigna/metabolismoRESUMEN
Amphiphilic block polypeptides of poly(sarcosine)-b-(l-Val-Aib)6 and poly(sarcosine)-b-(l-Leu-Aib)6 and their stereoisomers were self-assembled in water. Three kinds of binary systems of poly(sarcosine)-b-(l-Leu-Aib)6 with poly(sarcosine)-b-poly(d-Leu-Aib)6, poly(sarcosine)-b-poly(l-Val-Aib)6, or poly(sarcosine)-b-(d-Val-Aib)6 generated vesicles of ca. 200 nm diameter. The viscoelasticity of the vesicle membranes was evaluated by the nanoindentation method using AFM in water. The elasticity of the poly(sarcosine)-b-(l-Leu-Aib)6/poly(sarcosine)-b-poly(d-Leu-Aib)6 vesicle was 11-fold higher than that of the egg yolk liposome but decreased in combinations of the Leu- and Val-based amphiphilic polypeptides. The membrane elasticity is found to be adjustable by a suitable combination of helical blocks in terms of stereocomplex formation and the interdigitation of side chains among helices in the molecular assemblies.
Asunto(s)
Oligopéptidos/química , Elasticidad , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica , Sarcosina , Viscosidad , AguaRESUMEN
A cold-adapted bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid (EPA) as a component of its membrane phospholipids, is useful as a model to study the function of EPA and as a host for heterologous production of thermolabile proteins at low temperatures. In this study, we characterized extracellular membrane vesicles (EMVs) of this bacterium to examine the involvement of EPA in the biogenesis of EMVs and for the future application of EMVs to extracellular protein production. We found that this strain produced EMVs from the cell surface. Cryo-electron microscopic observation showed that the majority of the EMVs had a single-bilayer structure with an average diameter of 110 nm, though EMVs with double-bilayer membranes and other diverse structures were also observed. Quantitative analysis demonstrated that the EMV production was significantly increased (3-5 fold) by the depletion of EPA-containing phospholipids. The lack of EPA also altered the protein composition of EMVs. In particular, incorporation of one of the cold-inducible outer membrane proteins, OmpC176, was significantly increased in EMVs after the depletion of EPA. These results provide a basis for the construction of an EMV-based, low-temperature protein production system and show the involvement of EPA in the regulation of EMV biogenesis.
Asunto(s)
Fosfolípidos/metabolismo , Shewanella/metabolismo , Regiones Antárticas , Microscopía Electrónica de TransmisiónRESUMEN
Cyclic tri-ß-peptide having tetrathiafulvalene (TTF) at the side chain was synthesized to prepare a peptide nanotube aligning TTF side chains along the nanotube. The polarized light microscopic observations revealed crystallization of the cyclic peptide by the vapor diffusion method. Fourier-transform infrared and electron diffraction measurements of the crystals clarified formation of homogeneous hydrogen bonds making a columnar structure with a layer spacing of 4.9 Å. Electronic measurements of the peptide crystals on a gold mica substrate were carried out by the current sensing AFM. The current-voltage curves showed a rectification behavior, whose profile was consistent with a metal and p-type semiconductor junction. The p-type property is supported by the first principle calculations, which showed the HOMO orbital delocalizing fully over the plane of the TTF ring with the energy level of -5.1 eV. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 275-282, 2016.
Asunto(s)
Electrones , Compuestos Heterocíclicos/química , Nanotubos de Péptidos/química , Semiconductores , Cristalización , Enlace de Hidrógeno , Microscopía de Fuerza Atómica , Modelos Moleculares , Estructura Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Formosan subterranean termites, Coptotermes formosanus, tend to avoid pathogen odors when tested in Y-tube olfactometers, but approach and groom exposed nestmates to remove pathogens from their cuticle and maintain a healthy population. To better understand their differential reaction to pathogens and their odors, the relationship between odor cues and direction of motion was examined with the fungus Isaria fumosorosea K3 strain. The results indicate that nestmate odor was strongly attractive only in tests where fungal odors were present in both branches of the olfactometer. Termites generally avoid fungal odors when offered a choice without fungal odor. We also tested termite aversion to 3-octanone and 1-octen-3-ol, major surface chemical compounds of I. fumosorosea K3, and estimated the total mass of these compounds present on the conidial surface by direct extraction method. The total quantity of these chemicals on the surface of fungal conidia was estimated to be approximately 0.01 ng per 10(7) conidia. This study demonstrates a context dependent behavioral change in termites in response to the odors of pathogenic fungi.
Asunto(s)
Hypocreales/fisiología , Isópteros/microbiología , Isópteros/fisiología , Percepción Olfatoria , Animales , Señales (Psicología) , Locomoción , Odorantes/análisis , OrientaciónRESUMEN
BACKGROUND: Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition. RESULTS: A novel endotransglucosylase catalyzes cleavage and transfer of ß-glucans and decreases the physical strength of plant cell walls. CONCLUSION: Endotransglucosylation causes depolymerization and polymerization of ß-glucans, depending on substrate molecular size. SIGNIFICANCE: Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-ß-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing ß-1,4-linkages, in which 1,3-1,4-ß-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into ß-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of ß-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-ß-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize ß-glucans, depending on the size of the acceptor substrate.
Asunto(s)
Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Magnaporthe/enzimología , beta-Glucanos/metabolismo , Conformación de Carbohidratos , Pared Celular/química , Celulosa/biosíntesis , Clonación Molecular , Cotiledón/química , Cotiledón/citología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hidrólisis , Oligosacáridos/química , Oryza/microbiología , Hojas de la Planta/microbiología , Especificidad por Sustrato , Transcripción Genética , Triticum/química , Triticum/citologíaRESUMEN
pH-Responsive molecular assemblies with a variation in morphology ranging from a twisted ribbon, a helical ribbon, to a nanotube were prepared from a novel A3B-type amphiphilic peptide having three hydrophilic poly(sarcosine) (A block) chains, a hydrophobic helical dodecapeptide (B block), and two histidine (His) residues between the A3 and B blocks. The A3B-type peptide adopted morphologies of the twisted ribbon at pH 3.0, the helical ribbon at pH 5.0, and the nanotube at pH 7.4, depending upon the protonation states of the two His residues. On the other hand, another A3B-type peptide having one His residue between the A3 and B blocks showed a morphology change only between the helical ribbon and the relatively planar sheets with pH variation in this range. The morphology change is thus induced by one- or two-charge generation at the linking site of the hydrophilic and hydrophobic blocks of the component amphiphiles but in different ways.
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Histidina/química , Nanotubos/química , Péptidos/química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Electrónica de Transmisión , Nanotubos/ultraestructura , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
Unsymmetrical vesicular membranes were prepared from a binary mixture of the A3B-type and the AB-type host polypeptides, which were composed of the hydrophilic block (A) and the hydrophobic helical block (B) but with a different helix sense between the two host polypeptides. TEM and DLS revealed the formation of vesicles with ca. 100 nm diameter. The molecular assembly was driven by hydrophobic interaction, stereocomplex formation, and dipole-dipole interaction between hydrophobic helices. Furthermore, the A3B-type host polypeptide extended the hydrophilic block to the outer surface of vesicles as a result of the steric effect, resulting in the formation of unsymmetrical vesicular membranes. As a result, a functionalized AB-type guest polypeptide having the same helix sense with the A3B-type host polypeptide exposed the hydrophilic block to the outer surface. In contrast, an AB-type guest polypeptide having the same helix sense with the AB-type host polypeptide oriented the hydrophilic block to the inner surface. Functionalization of either the outer or inner surface of the binary vesicle is therefore facile to achieve when using either the right- or the left-handed helix of the functionalized guest polypeptide.
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Péptidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Secundaria de ProteínaRESUMEN
Cellulose is a high molecular weight polysaccharide of ß1 â 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.