RESUMO
We have recently shown that a dominant-negative mutant of CXCL8, dnCXCL8, with increased glycosaminoglycan (GAG) binding affinity and inactivated GPCR signaling function is able to efficiently prevent neutrophil infiltration into murine lungs (Adage et al., 2015). Here we present evidence that chemical PEGylation of dnCXCL8 with 20 kDa and 40 kDa PEG does not significantly interfere with GAG binding affinity, nor does it influence the mutant's disabled chemotaxis function, while it strongly improved bioavailability and serum half-life of the chemokine mutant. In a murine model of lung inflammation, only the 40 kDa PEGylated dnCXCL8 showed a significant reduction of neutrophils in bronchoalveolar lavage (BAL) fluid. In combination with an almost three-fold increase (compared to non-PEGylated dnCXCL8) in plasma half-life after intravenous administration, our results prove that PEGylation of chemokine-derived biologics is an amenable way for the treatment of chronic inflammatory conditions.
Assuntos
Glicosaminoglicanos/metabolismo , Interleucina-8/metabolismo , Mutação , Polietilenoglicóis/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Heparitina Sulfato/metabolismo , Humanos , Interleucina-8/genética , Interleucina-8/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Neutrófilos/citologia , Neutrófilos/metabolismo , Pneumonia/metabolismo , Ligação ProteicaRESUMO
The fluorescence of the rare amino acid LL-dityrosine, which is found in insoluble biological materials with structural features, was recently shown to decay non-exponentially (Kungl et al. (1992) J. Fluorescence 2, 63-74). Here we investigated the time-resolved fluorescence of a dityrosine-containing peptide (DCP) to study the influence of side chains on the fluorescence decay of the chromophore. The fluorescence decay of DCP was best fitted by three exponential terms including a sub-nanosecond rise term, the values of which are quite similar to the parameters obtained for the decay of free dityrosine. They were found to depend on the pH of the aqueous solution but not on the temperature. Analysis by an exponential series method revealed broad fluorescence lifetime distributions for DCP. Compared to the corresponding analysis of dityrosine transients, similar lifetime centers were found whereas the widths of the distributions were found broader for DCP. Molecular dyamics (MD) simulations of dityrosine at 300 K show that chi 1 and chi 2 side chain conformers (rotamers) of both tyrosine subunits interconvert on a picosecond timescale. The rates of interconversion were shown to depend critically upon the MD technique applied: in vacuo simulations yielded lower interconversion rates compared to stochastic dynamics (SD) and full MD (water explicitly included). However, MD simulations of the dityrosine-containing peptide revealed no interconversions of the chi 1 and chi 2 side chain rotamers of both tyrosine subunits within a 400 ps trajectory. Interconversions could be induced by raising the temperature of the system (DCP plus solvent) to 340 K. Side chain rotamers of dityrosine are not stable on a fluorescence time scale but are stable when a dityrosine-containing peptide is regarded. Nevertheless both molecules yield similar fluorescence decay patterns. We therefore conclude that the rotamer model proposed for the fluorescence decay of tyrosine and tryptophan cannot be applied to the fluorescence decay of dityrosine and peptides containing this chromophore. This should be of future interest when dityrosine is used as an intrinsic sensor to study complex dityrosine-containing macromolecules by fluorescence spectroscopy.
Assuntos
Peptídeos/química , Tirosina/análogos & derivados , Conformação Molecular , Espectrometria de Fluorescência , Tirosina/químicaRESUMO
Based on the X-ray structure of the human immunodeficiency virus type-1 (HIV-1) protease in complex with the statine-derived inhibitor SDZ283-910, a 542 ps molecular dynamics trajectory was computed. For comparison with the 805 ps trajectory obtained for the uncomplexed enzyme, the theoretical fluorescence anisotropy decay of the unliganded protease and the inhibitor complex was calculated from the trajectories of the Trp6A/Trp6B and Trp42A/Trp42B transition dipole moments. This enabled us to directly compare the simulated data with the experimental picosecond time-resolved fluorescence data. Fitting both experimental and simulated data to the Kohlrausch-Williams-Watts (KWW) function exp(-t/tauk)beta revealed a very good agreement for the uncomplexed protease as well as for the SDZ283-910 complex. Binding of the inhibitor induced a faster decay of both the experimental and the computed protease fluorescence anisotropy decay. By this integrative approach, the atomic detail of inhibitor-induced changes in the conformational dynamics of the HIV-1 protease was experimentally verified and will be used for further inhibitor optimisation.
Assuntos
Fármacos Anti-HIV/química , Inibidores da Protease de HIV/química , Protease de HIV/química , Oligopeptídeos/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/metabolismo , Simulação por Computador , Cristalografia por Raios X , Polarização de Fluorescência , Protease de HIV/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/metabolismo , HIV-1/enzimologia , Substâncias Macromoleculares , Modelos Moleculares , Conformação Proteica , Triptofano/químicaRESUMO
A new fluorescence method is introduced in which nitric oxide (NO)-derived higher-order oxygen complexes (NO(x)) are quantified at physiological pH. Detecting the fluorescence lifetime shift between 2,3-diaminonaphthalene and the NO(x)-derived protonated 2,3-naphthotriazole allows an intensity independent determination of the NO(x) concentration. The NO release from LPS and IFNgamma-stimulated murine macrophages and iNOS transfected hamster cells was quantified. The lower detection limit for NO2- was found to be 800 pmol/ml. Since the influence of static fluorescence quenching due to cellular components can be neglected, the method is applicable for clear cellular supernatants as well as turbid cellular suspensions.
Assuntos
Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/análise , 2-Naftilamina/análogos & derivados , Animais , Linhagem Celular , Cricetinae , Cricetulus , Indução Enzimática , Corantes Fluorescentes , Humanos , Interferon gama/farmacologia , Cinética , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Óxido Nítrico Sintase/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos , TransfecçãoRESUMO
The binding of aromatic amino acids to the ligand response domain of the tyrosine repressor (TyrR) protein (TyrR(lrd)) of Haemophilus influenzae was investigated using circular dichroism and fluorescence spectroscopy. The induced secondary structural changes were unique for each aromatic amino acid and were further influenced by the presence or absence of ATP. Tyrosine was found to have the highest affinity for TyrR(lrd) in the absence of ATP, whereas the affinity for ATP itself increased in the presence of tyrosine. Binding of tyrosine is therefore the conformational trigger for the activation of TyrR whereas ATP is regarded as a conformational co-activator.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Escherichia coli , Haemophilus influenzae/química , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Tirosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Dicroísmo Circular , Sequência Conservada , Ligantes , Fenilalanina/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Termodinâmica , Triptofano/metabolismo , Tirosina/agonistasRESUMO
The potent activator and chemoattractant for human neutrophils, neutrophil-activating peptide 2 (NAP-2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP-2 (rhNAP-2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP-2. rhNAP-2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 A, b = 39.5 A and c = 95.3 A. A packing density of 3.8 A3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 A on a conventional X-ray source. They are stable to X-rays for several days and are thus suitable for high resolution structure determination.
Assuntos
Cristalografia por Raios X , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cristalização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Humanos , Elastase de Leucócito , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Elastase Pancreática/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , beta-TromboglobulinaRESUMO
We investigated in vivo effects of norepinephrine (NE) on the transcription of 200 immunologically relevant genes in the mouse. Balb/c mice were s.c. implanted with NE containing retard tablets. Twelve hours later, splenic mRNA was prepared and hybridized onto cDNA microarrays containing the sequences of the major cytokines, their receptors and all CD-antigens of the mouse. Consistent results were obtained with a set of five genes: in the NE-treated animals four genes (CXCR4, VCAM1, IL-1R2, CD 14) were found 2-8 fold upregulated as compared to sham treated animals, whereas the gene for CCR3 was downregulated (< 0.5 fold). The findings were confirmed using quantitative reverse transcriptase Real Time PCR. These first results prove the usefulness of gene microarray technology towards transcription pattern analysis in neuroimmune interactions. Furthermore, they support the relevance of catecholamines in the regulation of leukocyte migration and the inflammatory response.
Assuntos
Quimiotaxia de Leucócito/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neuroimunomodulação/genética , Sistemas Neurossecretores/imunologia , Norepinefrina/farmacologia , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Receptores de Lipopolissacarídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/imunologia , Sistemas Neurossecretores/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores CCR3 , Receptores CXCR4/genética , Receptores de Quimiocinas/genética , Receptores de Interleucina-1/genética , Receptores Tipo II de Interleucina-1 , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Estresse Fisiológico/fisiopatologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Ativação Transcricional/imunologia , Regulação para Cima/genética , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
To test the hypothesis of charge-transfer quenching between an electrophile in the alanyl sidechain (carbonyl carbon or protonated amino group) and the excited aromatic phenol-subunit, which leads to a bi-exponential fluorescence decay of tyrosine in acidic aqueous solution, we investigated the dynamics of this amino acid and of the peptide Gly-Tyr-Gly in vacuo and in water with classical molecular dynamics (MD) and with stochastic dynamics (SD) computer simulation. The proposed low-frequency of interconversions between sidechain rotamers on a fluorescence time-scale could not be confirmed. Instead, frequent transitions for both, chi 1 and chi 2, dihedrals were observed. Simulating a low pH situation (protonated carboxylate group) did not significantly affect the transition frequency. Rotamer interconversions in the peptide Gly-Tyr-Gly, though significantly less, were also observed although the fluorescence decay of this compound could be described by a uni-modal lifetime distribution centered at 0.8 ns. The results obtained from simulations in vacuo and in solution were critically compared with those of stochastic simulations. We found the stochastic simulation in a better agreement to full MD (water explicitly included), which is highly time consuming, whereas the in vacuo simulations clearly deviated from both. We conclude from our results that, since the rotamers do frequently interconvert within the fluorescence lifetime of tyrosine, their contribution to the non-exponential fluorescence decay should be negligible.
Assuntos
Oligopeptídeos/química , Tirosina/química , Simulação por Computador , Conformação Molecular , Conformação Proteica , Espectrometria de Fluorescência/métodos , Processos Estocásticos , Fatores de TempoRESUMO
Glycosylation is the most common form of post-translational modifications by which oligosaccharide side chains are covalently attached to specific residues of the core protein. Especially O-linked glycan structures like the glycosaminoglycans were found to contribute significantly to many (patho-)biological processes like inflammation, coagulation, cancer and viral infections. Glycans exert their function by interacting with proteins thereby changing the structure of the interacting proteins and consequently modulating their function. Given the complex nature of cell-surface and extracellular matrix glycan structures, this therapeutic site has been neglected for a long time, the only exception being the antithrombin III-glycan interaction which has been successfully targeted by unfractionated and low-molecular weight heparins for many decades. Due to the recent breakthrough in the '-ome' sciences, among them proteomics and glycomics, protein-glycan interactions became more amenable for therapeutic approaches so that novel inhibitors of this interaction are currently in preclinical and clinical studies. An overview of current approaches, their advantages and disadvantages, is given and the promising potential of pharmacologically interfering with protein-glycan interactions is highlighted here.
Assuntos
Desenho de Fármacos , Glicosaminoglicanos/antagonistas & inibidores , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoglicanas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Sítios de Ligação , Configuração de Carboidratos , Glicômica/métodos , Glicosaminoglicanos/metabolismo , Glicosilação , Heparina/análogos & derivados , Heparina/biossíntese , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Proteoglicanas/biossíntese , Proteoglicanas/química , Proteômica/métodos , Relação Estrutura-AtividadeRESUMO
The interaction of chemokines and GAGs (glycosaminoglycans) on endothelial surfaces is a crucial step for establishing a chemotactic gradient which leads to the functional presentation of chemokines to their GPCRs (G-protein-coupled receptors) and thus to activation of approaching leucocytes. Based on molecular modelling, biophysical investigations, cell-based and in vivo experiments, we have developed a novel concept for therapeutically interfering with chemokine-GAG interactions, namely dominant-negative chemokine mutants with improved GAG binding affinity and knocked-out GPCR activity. These recombinant proteins displace their wild-type chemokine counterparts from the natural proteoglycan co-receptors without being able to activate leucocytes via GPCRs. Our mutant chemokines therefore represent the first protein-based GAG antagonists with high therapeutic potential in inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Proteoglicanas/antagonistas & inibidores , Proteoglicanas/metabolismo , Receptores de Quimiocinas/genética , Proteínas Recombinantes/uso terapêutico , Deleção de Sequência , Motivos de Aminoácidos/genética , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Quimiocinas/química , Quimiocinas/uso terapêutico , Humanos , Mutação , Ligação Proteica , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/deficiência , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The fluorescence decay of 1-palmitoyl-2-diphenyl-hexatrienyl-propionyl-sn-glycero-3-phosphocholine and the respective ethanolamine derivative were determined in human erythrocyte ghost membranes by phase and modulation fluorometry. The obtained bimodal distribution decay model could independently be confirmed by pulse fluorometry and data analysis by the exponential series method. Distributional widths of labeled choline and ethanolamine phospholipids were different in ghost membranes but identical in phospholipid vesicles. Thus, it is concluded that both lipid classes containing the same fluorophore experience environments of different heterogeneity in the biological system.
Assuntos
Membrana Eritrocítica/química , Lipídeos de Membrana/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Corantes Fluorescentes , Humanos , Técnicas In Vitro , Bicamadas Lipídicas/química , Espectrometria de FluorescênciaRESUMO
The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.
Assuntos
Saccharomyces cerevisiae/química , Tirosina/análogos & derivados , Membrana Celular/química , Polarização de Fluorescência/métodos , Saccharomyces cerevisiae/fisiologia , Espectrometria de Fluorescência/métodos , Esporos Fúngicos/química , Fatores de Tempo , Tirosina/análiseRESUMO
We have investigated the time-resolved fluorescence properties ofLL-dityrosine in aqueous solution. Typically, three exponential components were needed to fit the fluorescence pattern adequately, with pure decay terms for the low-intensity, high-energy state (λem = nm) but with a pronounced subnanosecond rise phase for the predominant red-edge fluorescence (λem > 380). Dual fluorescence behavior is indicative of an intramolecular precursorsuccessor pair, i.e., a consecutive intramolecular excited-state reaction. We suggest that this reaction is a torsional motion of the (deprotonated) monoanion along the biphenolic bond. Analysis of the fluorescence anisotropy decay of dityrosine yielded two rotational correlation times, the longer of which is associated with a negative preexponential term. The increase with time in the horizontally polarized component of the intensity decay is interpreted as the result of an electronic rearrangement in the excited state when the successor form of dityrosine is generated. Lifetime distributions of experimental data were probed by an unbiased exponential series method which uses a Tikhonov-type regularization function. The procedure revealed three well-separated groups of lifetimes, the short-lived ensemble forming a formally negative distribution. A photophysical model is introduced which interprets the biexponential decay of dityrosine in terms of overlapping emission signals from the precursor and the successor molecule.
RESUMO
The secondary structure of the ligand response domain of the Haemophilus influenzae tyrosine repressor, TyrR(lrd), was investigated using CD spectroscopy which revealed 42.5% alpha-helix, 17.6% beta-sheet, and 39.9% loops. Quaternary structure analysis by fluorescence anisotropy showed that TyrR(lrd) is monomeric at a concentration of 100 nM to 2 microM but that the protein readily dimerizes in the presence of its natural ligand ATP. Equilibrium unfolding studies of TyrR(lrd) using guanidinium hydrochloride suggested a two-state model with no detectable stable intermediates. The unfolding transition monitored by CD spectroscopy was responsive to tyrosine and ATP resulting in a shift to higher denaturant concentrations in the presence of these ligands. Differential scanning calorimetry yielded melting temperatures, T(m), of 51.15 and 58.07 degrees C for the unliganded and for the ATP-liganded protein, respectively. ATP is thus proposed to be a major structural cofactor for the molecular architecture of TyrR(lrd).
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli , Haemophilus influenzae/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sítios de Ligação , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Dimerização , Polarização de Fluorescência , Guanidina , Cinética , Ligantes , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Termodinâmica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The intrinsic tryptophan fluorescence signal of the full-length nuclear receptor hVDR was used to directly determine the dissociation constants, K(d), of two ligands yielding K(d) = 32 nM for 1alpha,25(OH)(2)D(3) and K(d) = 322 nM for 25(OH)D(3). Ligand binding was accompanied by a conformational change in the alpha-helical part of hVDR as revealed by CD spectroscopy. In addition, the presence of calcitriol was found to be a necessary prerequisite for the homodimerisation of hVDR which was monitored using fluorescence anisotropy. We conclude that the observed ligand-induced structural change of hVDR is conditional for dimerisation of the protein.
Assuntos
Receptores de Calcitriol/química , Calcifediol/química , Calcifediol/farmacologia , Calcitriol/química , Dicroísmo Circular , Dimerização , Relação Dose-Resposta a Droga , Polarização de Fluorescência , Humanos , Ligantes , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Receptores de Calcitriol/fisiologia , Relação Estrutura-Atividade , Vitamina D/análogos & derivados , Vitamina D/química , Vitamina D/farmacologiaRESUMO
Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS), but its function is not fully understood. Specifically, it is unclear whether BH4 participates directly in electron transfer. We investigated the redox properties of BH4 and several other pteridines with cyclic voltammetry and Osteryoung square wave voltammetry. BH4 was oxidized at a potential of +0.27 V vs normal hydrogen electrode (NHE); the corresponding reductive signal after the reversal of the scan direction was very small. Instead, reduction occurred at a potential of -0.16 V vs NHE; there was no corresponding oxidative signal. These two transitions were interdependent, indicating that the reductive wave at -0.16 V represented the regeneration of BH4 from its product of oxidation at +0.27 V. Similar voltammograms were obtained with tetrahydroneopterin and 6,7-dimethyltetrahydropterin, both of which can substitute for BH4 in NOS catalysis. Completely different voltammograms were obtained with 7,8-dihydrobiopterin, sepiapterin, 2'-deoxysepiapterin, and autoxidized BH4. These 7,8-dihydropterins, which do not sustain NOS catalysis, were oxidized at much higher potentials (+0.82-1.04 V vs NHE), and appreciable reduction did not occur between +1.2 and -0.8 V, in line with the concept of a redox role for BH4 in NOS catalysis. However, the electrochemical properties of the potent pterin-site NOS inhibitor 4-amino-BH4 resembled those of BH4, whereas the active pterin cofactor 5-methyl-BH4 was not re-reduced after oxidation. We conclude that the 2-electron redox cycling of the pterin cofactor between BH4 and quinonoid dihydrobiopterin is not essential for NO synthesis. The data are consistent with 1-electron redox cycling between BH4 and the trihydrobiopterin radical BH3(*).
Assuntos
Biopterinas/análogos & derivados , Biopterinas/química , Eletroquímica , Óxido Nítrico Sintase/antagonistas & inibidores , Pteridinas/química , Antioxidantes/química , Biopterinas/metabolismo , Catálise , Condutividade Elétrica , Óxido Nítrico Sintase/metabolismo , Oxirredução , Pteridinas/metabolismoRESUMO
We have investigated the time-resolved fluorescence of diphenylhexatriene (DPH) covalently linked to phosphatidylcholine (PC) in palmitoyloleoylglycerophosphocholine (POPC) vesicles with special consideration of the comparison of two methods for distributional lifetime analysis: the exponential-series method (ESM) and the maximum-entropy method (MEM). Generally, both methods were found to reveal equivalent results on high-quality data. Different are the shapes of the recovered distributions (symmetry and width) as well as the time effort for the numerical analysis.
RESUMO
The fluorescence intensity of the two flavin prosthetic groups, FMN and FAD, in neuronal nitric oxide synthase (nNOS) was found to decay highly nonexponentially, being best described by four fluorescence lifetimes. This excited state heterogeneity is the result of multiple flavin quenching sites which are due to several flavin microenvironments created mainly by stacking with aromatic amino acids. Investigating nNOS in the absence of one or more of Ca2+/calmodulin, tetrahydrobiopterin, and heme revealed an influence of these cofactors on the microenvironments of the flavin prosthetic groups. Similar effects on the flavin rotational dynamics were found by analyzing the fluorescence anisotropy decay of the holo and of the different apo forms of nNOS. Since the tetrahydrobiopterin and the heme are located in the N-terminal oxygenase domain of nNOS, their effect on the flavins in the C-terminal reductase domain is explained by a folding back of the reductase domain onto the oxygenase domain. Thereby a domain-domain interface is created containing the FAD, FMN, heme, and tetrahydrobiopterin prosthetic groups which allows for efficient electron transfer during catalysis. The heme group, which is known to be essential for homodimerization of nNOS, was also found to be essential for the formation of the domain-domain interface.
Assuntos
Flavinas/química , Proteínas do Tecido Nervoso/química , Óxido Nítrico Sintase/química , Termodinâmica , Animais , Arginina/metabolismo , Biopterinas/análogos & derivados , Biopterinas/química , Encéfalo , Calmodulina/química , Dimerização , Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Polarização de Fluorescência/métodos , Heme , Holoenzimas/química , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Ligação Proteica , Conformação Proteica , Ratos , Espectrometria de Fluorescência/métodos , Especificidade por SubstratoRESUMO
Fluorescence spectroscopy has been applied to the single tryptophan-containing regulatory protein Rev of human immunodeficiency virus (HIV-1). The fluorescence emission was found to have a maximum at 336 nm which refers to a surrounding of the chromophore of intermediate polarity. Fluorescence transients recorded at the maximum of fluorescence were found to decay nonexponentially. A bimodal lifetime distribution is obtained from exponential series analysis (ESM) with centers at 1.7 and 4.5 ns. Two microenvironments for tryptophan are suggested to be responsible for the two lifetime distributions. No innerfilter effect occurred in a Rev solution up to a concentration of 40 µM. A data quality study of ESM analysis as function of collected counts in the peak channel maximum (CIM) showed that, for reliable reconvolution, at least 15,000 CIM are necessary. The widths of the two distributions are shown to be temperature dependent. The broadening of the lifetime distributions when the temperature is raised to 50°C is interpreted as extension of the number of conformational substates which do not interconvert on the fluorescence time scale. The thermal deactivation (temperature quenching) is reflected in a constant decrease in the center of the short-lived lifetime distribution.
RESUMO
Bet v 1 is the major birch pollen allergen and therefore the main cause of type I allergies observed in early spring. It is composed of 159 amino acid residues adding up to a molecular mass of 17 kDa. We determined the secondary structure and tertiary fold of full-length Bet v 1 by NMR spectroscopy. Two- and three-dimensional NMR measurements suggest that Bet v 1 is a globular monomer in solution with a high content of well defined secondary structure. Of the total of 159 residues, 135 could be sequentially assigned, using an improved assignment strategy based mainly on heteronuclear experiments. An improved strategy for structure calculation revealed three helices and two beta-sheets as major elements of secondary structure. The globular tertiary structure is mainly stabilized by two antiparallel beta-sheets. The two helices at the C terminus are in accordance with the results from the solution structure of the chemically synthesized peptide Bet v 1-(125-154). This peptide is composed of two helices connected by a hinge. The structural features of Bet v 1 are highly similar to those found in the Ambrosia allergen Amb t V.