RESUMO
The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg(2+) ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites. Consequently, at low Mg(2+) concentrations, Mg(2+) binds preferentially to the open state, favoring its formation, but at high concentrations, Mg(2+) binds preferentially to the compact state. The affinity of the riboswitch for SAM increases drastically with an increased level of binding of Mg(2+) to the compact pseudoknot conformation. The effect of increasing concentrations of trimethylamine N-oxide (TMAO), a well-studied molecular crowding agent, on the conformation of the riboswitch and its affinity for SAM were also monitored by CD spectroscopy and measurement of diffusion. In the absence of added Mg(2+), high concentrations of TMAO were found to induce a conformational change compatible with the formation of the pseudoknot form but have only a small effect on the affinity of the RNA for SAM.
Assuntos
Magnésio/química , Metilaminas/química , Riboswitch , S-Adenosilmetionina/química , Quelantes/química , Dicroísmo Circular , Conformação ProteicaRESUMO
The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.
Assuntos
Aciltransferases/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Aciltransferases/química , Aciltransferases/genética , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise , Sequência de Carboidratos , Domínio Catalítico , Parede Celular/enzimologia , Parede Celular/metabolismo , Fatores Corda/metabolismo , Galactanos/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
BACKGROUND: Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin. METHODS AND FINDINGS: Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2-15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2ß1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K(D) 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease. CONCLUSION: Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.
Assuntos
Proteínas de Insetos/metabolismo , Inibidores da Agregação Plaquetária/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glândulas Salivares/química , Simuliidae/metabolismo , Sequência de Aminoácidos , Animais , Trombose das Artérias Carótidas/prevenção & controle , Colágeno Tipo III/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Adesividade Plaquetária , Agregação Plaquetária , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismoRESUMO
Development of simple processes to fractionate synthetic mixtures of single-wall carbon nanotubes (SWCNTs) into individual species is crucial to many applications. Existing methods for single-chirality SWCNT purification are cumbersome, often requiring multiple steps and different conditions for different species. Here, we report a method to achieve total fractionation of a synthetic SWCNT mixture by countercurrent chromatography, resulting in purification of many single-chirality SWCNT species in a single run. This method is based on a tunable partition of sodium deoxycholate dispersed SWCNTs in a polyethylene glycol/dextran aqueous two-phase system. By running the mobile phase with 0.02% of sodium deoxycholate and a gradient of sodium dodecyl sulfate from 0.1% to 0.7% (w/w), we observe clear diameter-dependent elution, with â¼ 90% total recovery. Among all the fractions collected, a number of them are enriched in single-chirality (9,4), (7,5), (7,6), (8,3), (6,5) species, while most of the remaining ones contain no more than 2-3 major species. We also observe strong (n,m)-dependent elution peak width due to the enantiomer-resolved partition. These results demonstrate countercurrent chromatography (CCC) as an effective way to obtain high purity (n, m) species, and suggest the potential of CCC as an analytical tool for chirality distribution mapping of synthetic SWCNT mixtures.
Assuntos
Distribuição Contracorrente/métodos , Nanotubos de Carbono/química , Ácido Desoxicólico/química , Dextranos , Planejamento Ambiental , Polietilenoglicóis , Dodecilsulfato de Sódio , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% ß-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.
Assuntos
Inibidores da Ornitina Descarboxilase , Ornitina Descarboxilase/química , Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Dicroísmo Circular , Escherichia coli/genética , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/isolamento & purificação , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH â¼5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel ß-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aß peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.
Assuntos
Amiloide/metabolismo , Antígeno gp100 de Melanoma/metabolismo , Amiloide/química , Amiloide/genética , Animais , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Peixe-Zebra , Antígeno gp100 de Melanoma/química , Antígeno gp100 de Melanoma/genéticaRESUMO
A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P-jump kinetics in the presence of Na(+), NH(4) (+), and Na(+) together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (beta(double dagger) (o)) term to obtain a good fit. beta(double dagger) (o) is positive for the Na(+) enzyme but negative for NH(4) (+) and Na(+) with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P-jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.
Assuntos
Pressão Hidrostática , Salmonella typhimurium/enzimologia , Triptofano Sintase/química , Regulação Alostérica , Cátions , Cinética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Salmonella typhimurium/genética , Serina/química , Solventes , Espectrometria de Fluorescência , Espectrofotometria , Termodinâmica , Triptofano Sintase/genética , Triptofano Sintase/metabolismoRESUMO
Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light-scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography. Surface plasmon resonance identified the sequence RGQOGVMGF (where O is hydroxyproline) that mediates collagen interaction with von Willebrand factor (vWF) as a high-affinity binding site for aegyptin, with a K(D) of approximately 5 nM. Additionally, aegyptin interacts with the linear peptide RGQPGVMGF and heat-denatured collagen, indicating that the triple helix and hydroxyproline are not a prerequisite for binding. However, aegyptin does not interact with scrambled RGQPGVMGF peptide. Aegyptin also recognizes the peptides (GPO)(10) and GFOGER with low affinity (microM range), which respectively represent glycoprotein VI and integrin alpha2beta1 binding sites in collagen. Truncated forms of aegyptin were engineered, and the C-terminus fragment was shown to interact with collagen and to attenuate platelet aggregation. In addition, aegyptin prevents laser-induced carotid thrombus formation in the presence of Rose Bengal in vivo, without significant bleeding in rats. In conclusion, aegyptin interacts with distinct binding sites in collagen, and is useful tool to inhibit platelet-collagen interaction in vitro and in vivo.
Assuntos
Trombose das Artérias Carótidas/prevenção & controle , Colágeno/química , Colágeno/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Proteínas e Peptídeos Salivares/metabolismo , Proteínas e Peptídeos Salivares/farmacologia , Fator de von Willebrand/metabolismo , Aedes/genética , Aedes/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Fenômenos Biofísicos , Colágeno/genética , DNA Complementar/genética , Feminino , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Hidroxiprolina/química , Técnicas In Vitro , Proteínas de Insetos/química , Proteínas de Insetos/genética , Integrina alfa2beta1/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Ratos , Ratos Wistar , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solid-state NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.
Assuntos
Amiloide/química , Melaninas/química , Melanossomas/metabolismo , Glicoproteínas de Membrana/química , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Melanócitos/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Antígeno gp100 de MelanomaRESUMO
The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-beta structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel beta-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a beta-helix-like structure but are not sufficient to establish such a structure definitively.
Assuntos
Amiloide/química , Proteínas de Escherichia coli/química , Sequência de Aminoácidos , Benzotiazóis , Biofilmes , Dicroísmo Circular , Endopeptidase K/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Príons/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Tiazóis/químicaRESUMO
Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.
Assuntos
Temperatura Alta , Salmonella typhimurium/enzimologia , Triptofano Sintase/química , Triptofano Sintase/metabolismo , Ativação Enzimática , Cinética , Estrutura Molecular , Pressão , Ligação Proteica , Serina/metabolismoRESUMO
The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to beta(1-3)glucose branches of beta(1-6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to beta(1-6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Parede Celular/química , Quitina/química , Quitinases/química , Digitonina/farmacologia , Corantes Fluorescentes/química , Glicosilação , Técnicas In Vitro , Microscopia de Fluorescência/métodos , Modelos Biológicos , Modelos Genéticos , Oligossacarídeos/química , Permeabilidade , Fatores de TempoRESUMO
Caliciviruses have a positive strand RNA genome covalently-linked at the 5'-end to a small protein, VPg. This study examined the biochemical modification of VPg by the ProPol form of the polymerase of human norovirus strain MD145 (GII.4). Recombinant norovirus VPg was shown to be nucleotidylylated in the presence of Mn2+ by MD145 ProPol. Phosphodiesterase I treatment of the nucleotidylylated VPg released the incorporated UMP, which was consistent with linkage of RNA to VPg via a phosphodiester bond. Mutagenesis analysis of VPg identified Tyrosine 27 as the target amino acid for this linkage, and suggested that VPg conformation was important for the reaction. Nucleotidylylation was inefficient in the presence of Mg2+; however the addition of full- and subgenomic-length MD145 RNA transcripts led to a marked enhancement of the nucleotidylylation efficiency in the presence of this divalent cation. Furthermore, evidence was found for the presence of an RNA element near the 3'-end of the polyadenylated genome that enhanced the efficiency of nucleotidylylation in the presence of Mg2+.
Assuntos
Produtos do Gene pol/metabolismo , Norovirus/metabolismo , Uridina Monofosfato/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Coenzimas/farmacologia , Ativadores de Enzimas/farmacologia , Produtos do Gene pol/isolamento & purificação , Magnésio/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Norovirus/enzimologia , Fosfodiesterase I/metabolismo , RNA Mensageiro/farmacologia , RNA Viral/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tirosina/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Estimation of a protein's secondary structure from its circular dichroism spectrum usually requires accurate knowledge of the concentration and pathlength of the sample. Two recently described methods avoid this problem by analysis of g-factor spectra (McPhie, Anal. Biochem. 293, 109-119) or scaling of relative intensities (Raussens et al., Anal. Biochem. 319, 114-121). Application of the two methods to the same samples shows that they can have similar efficacies. Calculation with the latter method is more rapid, but the performance of the former is maintained over reduced wavelength ranges.
Assuntos
Dicroísmo Circular , Proteínas/química , Estrutura Secundária de ProteínaRESUMO
Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase alpha2beta2 multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium alpha-glycerophosphate (Na2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of betaE109D and betaD305A mutations, on K(eq) for the conformational equilibria were determined. The l-Ser external aldimine-aminoacrylate equilibrium (K(eq)=[external aldimine]/[aminoacrylate]) has the largest value with Na+ (0.12), followed by K+ (0.04), Li+ (7.6 x 10(-4)), Rb+ (4.3 x 10(-4)), NH4+ (2.3 x 10(-4)), no cation (2.0 x 10(-4)) and Cs+ (1.6x10(-5)). alpha-Site ligands, Na(2)GP and IAG, have modest 3- to 40-fold effects on K(eq) in the direction of aminoacrylate, but BZI in the presence of Na+ gives a low value of K(eq) comparable to that obtained with Cs(+). There is no additivity of free energy for Na2GP and BZI, suggesting a common pathway for allosteric communications for both ligands. The values of DeltaV(o) range from -126 mL/mol for the Na+ complex to -204 mL/mol for the Na+ complex with BZI. The betaD305A mutation changes the K(eq) by a factor of at least 10(5) (26.7kJ/mol) and nearly abolishes allosteric communications. There are also dramatic decreases in the magnitude of both DeltaV(o) and DeltaS for the l-Ser external aldimine-aminoacrylate equilibrium for betaD305A Trp synthase, consistent with a large decrease in solvation accompanying the conformational change in betaD305A Trp synthase relative to wild-type Trp synthase. The betaE109D mutation has more modest but significant effects on K(eq), which differ with the ligand, ranging from 40-fold for GP to 2200-fold for BZI, even though betaGlu-109 is not directly involved in allosteric communications. The effect of GP on the external aldimine-quinonoid intermediate equilibrium of the Trp synthase-l-Trp complex is similar to that of GP on the Trp synthase-l-Ser external aldimine-aminoacrylate equilibrium. These results have allowed a quantitative comparison of the allosteric effects of ligand and mutations in Trp synthase. These allosteric effects are finely tuned to control the synthesis of l-Trp without resulting in substrate or product inhibition.
Assuntos
Salmonella typhimurium/enzimologia , Triptofano Sintase/química , Triptofano Sintase/ultraestrutura , Ativação Enzimática , Isomerismo , Ligantes , Mutação , Relação Estrutura-AtividadeRESUMO
Ag presentation is a key step in the initiation of adaptive immune responses that depends on the expression of MHC Ags and costimulatory molecules. Immune-enhancing CpG and non-CPG oligodeoxynucleotides (ODNs) stimulate Ag presentation by stimulating the expression of these molecules and by promoting dendritic cell maturation. In this report, we identify immunoregulatory orthophosphorothioate non-CpG molecules, referred to as regulatory ODNs (rODNs), by their ability to inhibit allogeneic monocyte-stimulated T cell responses and down-regulate HLA-DR in human primary monocytes. The rODNs promoted the survival of macrophages and were able to activate IL-8 secretion through a chloroquine-resistant pathway. Messenger RNAs for HLA-DR alpha and beta and the MHC CIITA were reduced by rODNs but not by stimulatory CpG ODN2006 and non-CpG ODN2006a. CIITA transcription in monocytes was controlled primarily by promoter III and not by promoter I or IV. rODNs blocked promoter III-directed transcription of CIITA in these cells. Under conditions that induced dendritic cell differentiation, rODNs also reduced HLA-DR expression. The activity of rODNs is phosphorothioate chemistry and G stretch dependent but TLR9 independent. G tetrads were detected by circular dichroism in active rODNs and associated with high m.w. multimers on nondenaturing gels. Heat treatment of rODNs disrupted G tetrads, the high m.w. aggregates, and the HLA-DR inhibitory activity of the ODNs. The inhibition of immune responses by regulatory oligodeoxynucleotides may be useful for the treatment of immune-mediated disorders including autoimmune diseases and graft rejection.
Assuntos
Adjuvantes Imunológicos/farmacologia , Regulação para Baixo/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Oligodesoxirribonucleotídeos/farmacologia , Regiões Promotoras Genéticas/imunologia , Transativadores/antagonistas & inibidores , Transativadores/genética , Proliferação de Células , Células Cultivadas , Inibidores do Crescimento/fisiologia , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/metabolismo , Cadeias alfa de HLA-DR , Humanos , Isoantígenos/farmacologia , Proteínas Nucleares/biossíntese , Regiões Promotoras Genéticas/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Transativadores/biossíntese , Transcrição Gênica/imunologiaRESUMO
At pH 2 apomyoglobin is extensively unfolded. Addition of increasing concentration of salts has been shown to convert the protein into molten globule form(s), which can undergo both heat-induced and cold-induced unfolding. Increasing concentrations of an inert polymer, dextran, lead to increased formation of molten globule and stabilizes the protein with respect to both heat-induced and cold-induced denaturation. The transitions were studied by circular dichroism. Two-state analysis of the data shows that the effects of salt and polymer are additive, and that stabilization by the polymer is independent of temperature, as predicted by excluded volume theory.
Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Temperatura Baixa , Temperatura Alta , Mioglobina/química , Mioglobina/metabolismo , Dobramento de Proteína , Cinética , Desnaturação Proteica/fisiologiaRESUMO
Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.
Assuntos
Ferro/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação ProteicaRESUMO
Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.