RESUMEN
Oligodendrocyte gene expression is downregulated in stress-related neuropsychiatric disorders, including depression. In mice, chronic social stress (CSS) leads to depression-relevant changes in brain and emotional behavior, and the present study shows the involvement of oligodendrocytes in this model. In C57BL/6 (BL/6) mice, RNA-sequencing (RNA-Seq) was conducted with prefrontal cortex, amygdala and hippocampus from CSS and controls; a gene enrichment database for neurons, astrocytes and oligodendrocytes was used to identify cell origin of deregulated genes, and cell deconvolution was applied. To assess the potential causal contribution of reduced oligodendrocyte gene expression to CSS effects, mice heterozygous for the oligodendrocyte gene cyclic nucleotide phosphodiesterase (Cnp1) on a BL/6 background were studied; a 2 genotype (wildtype, Cnp1+/- ) × 2 environment (control, CSS) design was used to investigate effects on emotional behavior and amygdala microglia. In BL/6 mice, in prefrontal cortex and amygdala tissue comprising gray and white matter, CSS downregulated expression of multiple oligodendroycte genes encoding myelin and myelin-axon-integrity proteins, and cell deconvolution identified a lower proportion of oligodendrocytes in amygdala. Quantification of oligodendrocyte proteins in amygdala gray matter did not yield evidence for reduced translation, suggesting that CSS impacts primarily on white matter oligodendrocytes or the myelin transcriptome. In Cnp1 mice, social interaction was reduced by CSS in Cnp1+/- mice specifically; using ionized calcium-binding adaptor molecule 1 (IBA1) expression, microglia activity was increased additively by Cnp1+/- and CSS in amygdala gray and white matter. This study provides back-translational evidence that oligodendrocyte changes are relevant to the pathophysiology and potentially the treatment of stress-related neuropsychiatric disorders.
Asunto(s)
Oligodendroglía/metabolismo , Conducta Social , Estrés Psicológico/genética , Transcriptoma , Amígdala del Cerebelo/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Corteza Prefrontal/metabolismo , Estrés Psicológico/metabolismoRESUMEN
Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain, is fundamental to brain function and implicated in the pathophysiology of several neuropsychiatric disorders. GABA activates G-protein-coupled GABAB receptors comprising principal GABAB1 and GABAB2 subunits as well as auxiliary KCTD8, 12, 12b and 16 subunits. The KCTD12 gene has been associated with bipolar disorder, major depressive disorder and schizophrenia. Here we compare Kctd12 null mutant (Kctd12(-/-)) and heterozygous (Kctd12(+/-)) with wild-type (WT) littermate mice to determine whether lack of or reduced KCTD12 expression leads to phenotypes that, extrapolating to human, could constitute endophenotypes for neuropsychiatric disorders with which KCTD12 is associated. Kctd12(-/-) mice exhibited increased fear learning but not increased memory of a discrete auditory-conditioned stimulus. Kctd12(+/-) mice showed increased activity during the inactive (light) phase of the circadian cycle relative to WT and Kctd12(-/-) mice. Electrophysiological recordings from hippocampal slices, a region of high Kctd12 expression, revealed an increased intrinsic excitability of pyramidal neurons in Kctd12(-/-) and Kctd12(+/-) mice. This is the first direct evidence for involvement of KCTD12 in determining phenotypes of emotionality, behavioral activity and neuronal excitability. This study provides empirical support for the polymorphism and expression evidence that KCTD12 confers risk for and is associated with neuropsychiatric disorders.
Asunto(s)
Conducta Animal , Emociones , Hipocampo/metabolismo , Aprendizaje , Receptores de GABA/genética , Animales , Ritmo Circadiano/genética , Miedo , Heterocigoto , Memoria , Ratones , Ratones Noqueados , Actividad MotoraRESUMEN
To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.
Asunto(s)
Glicoconjugados/metabolismo , Lectinas/metabolismo , Análisis por Micromatrices , Células CACO-2 , Glicoconjugados/química , Humanos , Lectinas/química , Estructura Molecular , Polisacáridos/química , Polisacáridos/metabolismoRESUMEN
The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Disacáridos/farmacología , Monosacáridos/farmacología , Poliestirenos/química , Animales , Sitios de Unión , Adhesión Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/metabolismo , Diazometano/química , Disacáridos/química , Disacáridos/metabolismo , Microanálisis por Sonda Electrónica , Galactosa/metabolismo , Galactosa/farmacología , Hepatocitos/química , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Lactosa/metabolismo , Lactosa/farmacología , Lectinas/metabolismo , Monosacáridos/química , Monosacáridos/metabolismo , Ratas , Sialiltransferasas/metabolismo , Espectrometría de Masa de Ion Secundario , Propiedades de SuperficieRESUMEN
Poly(dimethylsiloxane) (PDMS) appeared recently as a material of choice for rapid and accurate replication of polymer-based microfluidic networks. However, due to its hydrophobicity, the surface strongly interacts with apolar analytes or species containing apolar domains, resulting in significant uncontrolled adsorption on channel walls. This contribution describes the application and characterization of a PDMS surface treatment that considerably decreases adsorption of low and high molecular mass substances to channel walls while maintaining a modest cathodic electroosmotic flow. Channels are modified with a three-layer biotin-neutravidin sandwich coating, made of biotinylated IgG, neutravidin, and biotinylated dextran. By replacing biotinylated dextran with any biotinylated reagent, the modified surface can be readily patterned with biochemical probes, such as antibodies. Combination of probe immobilization chemistry with low nonspecific binding enables affinity binding assays within channel networks. The example of an electrokinetic driven, heterogeneous immunoreaction for human IgG is described.
Asunto(s)
Dimetilpolisiloxanos/química , Inmunoquímica/instrumentación , Espectrometría de Fluorescencia/instrumentación , Humanos , Inmunoglobulina G/análisis , MicrocomputadoresRESUMEN
In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-field-induced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.
Asunto(s)
Técnicas Biosensibles/métodos , Adsorción , Animales , Bovinos , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía Fluorescente , Fotoquímica , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Espectrometría de Masa de Ion Secundario , Propiedades de Superficie , TransductoresRESUMEN
Biological systems make considerable use of specific molecular interactions. Many biomolecules involved in biorecognition are glycosylated, the carbohydrate moiety playing an essential role. Controlled surface glycoengineering is thus of crucial importance in biosensing, cell guidance, and biomedical applications. This study describes the synthesis of an aryldiazirine-derivatized galactose and the functionalization of surfaces by carbohydrates using photochemical immobilization techniques. A photoactivatable glycosylated reagent was synthesized by addition of thiogalactopyranose to the maleimide group of N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide (MAD) to give N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-[3-thio (1-D-galactopyranosyl)succinimidyl]butyramide (MAD-Gal). The structure of the newly synthesized molecule was confirmed by UV spectroscopy, photoactivation, 1H NMR, and 13C NMR. MAD-Gal was immobilized on thin diamond films by photoactivation of the diazirine function (350 nm). Surface modification was investigated by XPS (X-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). Imaging ToF-SIMS was applied to detect glycopatterns generated by mask-assisted lithography.
Asunto(s)
Glicósidos/síntesis química , Glicósidos/química , Glicosilación , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Fotoquímica , Espectrometría de Masa de Ion SecundarioRESUMEN
A new method is presented for the covalent binding of dextran as an intermediate layer on surface acoustic wave (SAW) devices. For biosensing applications in aqueous media commercially available SAW devices require surface passivation to prevent corrosion of the aluminum device structures in electrolytes. Thin films of polyimide and parylene revealed exceptional passivation properties. They were used as a base for dextran immobilization. Covalent binding of dextran to polymer-coated surfaces was achieved by photoimmobilization. Aryldiazirine-functionalized bovine serum albumin served as the multifunctional light-activable linking agent (photolinker polymer). Dextran and photolinker polymer were mixed and photobonded to sensor surfaces. Essential photoimmobilization parameters were optimized. The binding of proteins to dextran applying carbodiimide chemistries was exemplified with antiurease antibodies and the feasibility of specific immunosensing was investigated on SAW sensors connected to a fluid handling system.
Asunto(s)
Técnicas Biosensibles , Acústica , Animales , Bovinos , Dextranos , Luz , PolímerosRESUMEN
Growing neurites are guided through their environment during development and regeneration via different cellular and extracellular matrix (ECM) molecular cues. To mimic cell-matrix interactions, a three-dimensional (3D) hydrogel-based ECM equivalent containing a covalently immobilized laminin oligopeptide sequence was designed to facilitate nerve regeneration. This study illustrates that the oligopeptide domain CDPGYIGSR covalently linked to an agarose gel as a bioartificial 3D substrate successfully supports neurite outgrowth from dorsal root ganglia (DRG) in vitro. The specificity of the neurite promoting activity was illustrated through the inhibition of neurite outgrowth from DRG in a CDPGYIGSR-derivatized gel in the presence of solubilized CDPGYIGSR peptide. Gels derivatized with CDPGYIGSK and CDPGRGSYI peptides stimulated a smaller increase of neurite outgrowth. In vivo experiments revealed the capability of a CDPGYIGSR-derivatized gel to enhance nerve regeneration in a transected rat dorsal root model compared to an underivatized gel, a CDPGRGSYI gel, and saline-filled nerve guidance channels. These data suggest the feasibility of a 3D hydrogel-based ECM equivalent capable of enhancing neurite outgrowth in vitro and in vivo.
Asunto(s)
Órganos Artificiales , Ingeniería Biomédica , Matriz Extracelular , Regeneración Nerviosa , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Embrión de Pollo , Ganglios Espinales/fisiología , Ganglios Espinales/ultraestructura , Geles , Masculino , Neuritas/fisiología , Neuritas/ultraestructura , Oligopéptidos/química , Ratas , Ratas Wistar , SefarosaRESUMEN
Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg-Gly-Asp-Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.
RESUMEN
A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde concentration in the outer monolayer.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Membranas Artificiales , Proteínas/metabolismo , Acilación , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Proteínas Bacterianas/metabolismo , Reactivos de Enlaces Cruzados , Cisteína/metabolismo , Vidrio , Maleimidas/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Óptica y Fotónica , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Silanos , Estreptavidina , Compuestos de Sulfhidrilo/metabolismoRESUMEN
To attain light-dependent functionalization of biocompatible materials, a photolabel-derivatized, bioactive laminin fragment has been synthesized, chemically characterized, and photoimmobilized. Covalent high-resolution patterning of the laminin fragment CDPGYIGSR to hydroxylated fluorinated ethylene propylene (FEP-OH), poly(vinyl alcohol), and glycophase glass has been achieved. The synthetic peptide CDPGYIGSR was thermochemically coupled to either N-[m-[3-(trifluoromethyl)-diazirin-3-yl]phenyl]-4-maleimidobuty ramide or 4-maleimidobenzophenone. Photolabel-derivatized peptides were radiolabeled, and 20 and 300 microns-sized patterns were visualized by autoradiography. The biospecific interaction of photoimmobilized laminin fragments with cells was investigated by analyzing the selective attachment of NG 108-15 neuroblastoma x glioma cells which bear CDPGYIGSR-specific cell surface receptors. On photopatterned FEP-OH membranes NG 108-15 cells differentiated in serum-supplemented media within 1 day. Specific attachment to the immobilized oligopeptide CDPGYIGSR was assessed in serum-free media with competitive binding studies, showing an 82% decrease in cell adherence after the cell receptors were blocked with soluble CDPGYIGSR.
Asunto(s)
Adhesión Celular , Células Inmovilizadas , Laminina , Neuronas/citología , Neuronas/fisiología , Fragmentos de Péptidos , Secuencia de Aminoácidos , Animales , Azirinas , Benzofenonas , Unión Competitiva , Vidrio , Glioma , Células Híbridas , Indicadores y Reactivos , Maleimidas , Datos de Secuencia Molecular , Estructura Molecular , Neuroblastoma , Fragmentos de Péptidos/síntesis química , Fotoquímica , Politetrafluoroetileno/análogos & derivados , Alcohol Polivinílico , Receptores de Superficie Celular/fisiologíaRESUMEN
Antibodies and antigen binding fragments thereof were photochemically immobilized on surface-modified silicon chips of 5 x 5 mm size. Silicon surface-grafted diazirines and benzophenones formed covalent bonds with the immunoreagents on light activation. Photolithographic immobilization of monoclonal antibodies in aqueous media was achieved on silicon chips by activating surface-grafted benzophenones. The presence of bovine serum albumin during irradiation reduced nonspecific adsorption of the immunoreagents and retained the immunoactivity of the photoimmobilized molecules.
Asunto(s)
Anticuerpos Monoclonales/química , Silicio/química , Animales , Ratones , Fotoquímica/métodosRESUMEN
Immunocomplexation at wave guiding TiO2/SiO2 surfaces was investigated using an integrated optical grating coupler. For extended application of this label-free monitoring system, F(ab')2 fragments of monoclonal antibodies were photo-immobilized by photolinker polymer-mediated procedures that do not require functionalization of either the immunoreagent or the TiO2/SiO2 surface. Covalent, light-dependent binding of photolinker polymer and F(ab')2 fragments was achieved using a single-step photo-reaction. Bovine serum albumin derivatized with aryldiazirines (T-BSA) served as a photolinker polymer. T-BSA suppressed the non-specific adsorption of analytes to wave guide surfaces. Immunoreagent binding and immunological activity were analyzed and modified surfaces were investigated by scanning force microscopy. Apparent immunoreagent surface densities were 16.7 fmol F(ab')2 per mm2 sensor surface. Optical analyses revealed linear, dose-dependent antigen binding with label free analytes. Immunocompetent surfaces were regenerable by treatment at pH 2.3, rendering the immunosensing system applicable for repetitive use.
Asunto(s)
Técnicas Biosensibles , Inmunoglobulinas/análisis , Reacciones Antígeno-Anticuerpo , FotoquímicaRESUMEN
Photolinker-polymer-mediated covalent immobilization of antibodies, F(ab') and F(ab')2 fragments has been achieved by light-dependent coupling procedures. Anti-alpha-foetoprotein (anti-AFP) monoclonal antibodies were covalently linked to microplates by layer-coating procedures, which entail antibody photoimmobilization to a photolinker-polymer-precoated surface. In this and the co-coating procedure described, diazirine-functionalized BSA (T-BSA) served as the multifunctional light-activatable linking agent (photolinker polymer). Prior to photo-activation, F(ab')2 or F(ab') fragments derived from anti-(prostate-specific antigen) monoclonal antibodies were mixed and co-coated with the photolinker polymer on to polystyrene microplates. The immunoreagents remained immunologically active after 350 nm irradiation (irradiance 0.7 mW.cm-2 for 20 min). Immuno-responses of photoimmobilized monoclonal anti-AFP antibodies were equivalent to signal intensities obtained with physically adsorbed antibodies. Photoimmobilization of anti-PSA F(ab') fragments in the presence of T-BSA revealed exponential binding characteristics indicating stabilizing molecular co-operativity of the BSA constituent. Co-coating procedures yielded 62 and 65% binding of applied 14C-labelled F(ab')2 and F(ab') fragments respectively. Covalency of antibody binding was inferred from: (i) the strict dependence of photoreagent availability; (ii) the light-dependence of the immobilization process; and (iii) the reversibility of immunocomplexation after acid treatment.
Asunto(s)
Anticuerpos Monoclonales , Azirinas/química , Proteínas Bacterianas/química , Fragmentos Fab de Inmunoglobulinas , Polímeros , Albúmina Sérica Bovina/química , Adsorción , Ensayo de Inmunoadsorción Enzimática , Fotoquímica , Estreptavidina , Radioisótopos de AzufreRESUMEN
Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.
Asunto(s)
Azirinas/síntesis química , Reactivos de Enlaces Cruzados/síntesis química , Maleimidas/síntesis química , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Aminoácidos/química , Anticuerpos Monoclonales/química , Azirinas/química , Radioisótopos de Carbono , Reactivos de Enlaces Cruzados/química , Cisteína/química , Vidrio , Fragmentos de Inmunoglobulinas/química , Maleimidas/química , Datos de Secuencia Molecular , Péptidos/química , Fotoquímica , Poliestirenos/química , Antígeno Prostático Específico/inmunología , Termodinámica , Tirosina/químicaRESUMEN
Purple membrane was regenerated from the denatured proteolytic (protease V8) fragments V-1 and V-2 of bacteriorhodopsin (BR), native membrane lipids, and all-trans-retinal. FTIR difference spectra of M and N intermediates of the reconstituted system are in close correspondence to those obtained from native BR. Asp-212 is the only internal aspartic acid in the V-2 fragment (helices F and G). Reconstituting a V-2 fragment from a [4-13C]Asp-labeled BR preparation with an unmodified V-1 fragment and vice versa have allowed us to assign IR bands to either Asp-212 or any of the remaining aspartic acids on V-1 (helices A-E). A carboxylate vibration at 1392 cm-1 has been identified in the M and N intermediates and assigned to Asp-212. Since no contribution of this residue to C = O stretches of protonated carboxyl groups was detected, Asp-212 must be ionized in light-adapted BR as well. The effect of [4-13C]Asp labeling of V-1 revealed a carboxylate vibration at 1385 cm-1 in light-adapted BR. Since Asp-96 and Asp-115 are protonated, this band is caused by Asp-85. All absorption changes of C = O stretches of protonated carboxyl groups are due to Asp residues on V-1. Correspondingly, the proton acceptor for Schiff base deprotonation in M is located on V-1, and must be Asp-85 (the only ionized Asp on V-1). The band assignments are compared with those reported for BR mutants, and the potential role of Asp-212 for proton translocation is discussed.
Asunto(s)
Ácido Aspártico/química , Bacteriorodopsinas/química , Espectrofotometría Infrarroja , Isótopos de Carbono , Análisis de Fourier , Marcaje Isotópico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fotoquímica , Bases de Schiff/química , Serina Endopeptidasas/metabolismoRESUMEN
We describe a novel, versatile procedure for the light-dependent immobilization of ligands to 'inert' material surfaces. Covalent immobilization of ligands differing in chemical nature and complexity is accomplished under mild and non-destructive conditions. Topical interaction of ligands with organic or inorganic surfaces is mediated by photoactivable polymers with carbene generating trifluoromethyl-aryl-diazirines which serve as linker molecules. Light activation of aryl-diazirino functions at 350 nm yields highly reactive carbenes, and covalent coupling is achieved by simultaneous carbene insertion into both the ligand and inert surface. Thus, reactive functional groups are not required on either the ligand or the supporting material. These procedures are applicable whenever ligands, from molecules to cells--synthetically or genetically produced, or isolated from biological sources--need to be immobilized for improved performance.
Asunto(s)
Azirinas/química , Biotecnología/métodos , Luz , Fosfatasa Alcalina/química , Proteínas Bacterianas/química , Ligandos , Fotoquímica , Poliestirenos , Polivinilos/química , Estreptavidina , Propiedades de SuperficieRESUMEN
A novel bilayer-forming phospholipid analogue with a photoactivatable carbene-generating head group was synthesized and characterized with respect to molecular structure and light-induced reactivity. N'-(1,2-Dimyristoyl-sn-glycero-3-phosphoethyl)-N-[m-[3- (trifluoromethyl)diazirin-3-yl]phenyl]thiourea (PED) was prepared by thiocarbamoylation of synthetic dimyristoylphosphatidylethanolamine with 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine. PED formed liposomes in aqueous media. Gel to liquid-crystalline transitions occurred at 10.5 degrees C. Neither PED- nor PED/dimyristoylphosphatidylcholine mixed liposomes underwent major structural changes when photoactivated. Liposome sizes, determined by electron microscopy, were not altered upon light exposure. PED combines the advantages of facile synthesis and timed carbene reactivity by photoactivation at wavelengths greater than or equal to 320 nm. Conditions used for PED photoactivation did not inactivate catalytically active or complex-forming proteins. Light-induced binding of aqueous-soluble proteins to PED containing liposomes was attained through photoactivation in the presence of myoglobin, streptavidin, or trypsin. The proteins mentioned were utilized to characterize carbene-initiated ligand coupling. Procedures described establish a new and versatile method for the formation of proteoliposomes.
Asunto(s)
Liposomas/química , Fosfolípidos/química , Proteínas/química , Proteínas Bacterianas/química , Rastreo Diferencial de Calorimetría , Cinética , Luz , Microscopía Electrónica , Mioglobina/química , Fotoquímica , Solubilidad , Estreptavidina , Tripsina/químicaRESUMEN
Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its alpha carbon (C alpha--H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the C alpha--H. The vibrational modes that exhibit this coupling are principally associated with C15--H and N--H vibrations. [C alpha--2H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the C alpha--2H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15--H out-of-plane wag is interpreted as a possible indication of a twist around the C15 = N bond.