RESUMEN
The kinetics of oxidation reactions of flavonoids, quercetin, dihydroquercetin, and epicatechin has been studied in the presence of biocatalysts of different natures: horseradish peroxidase, mushroom tyrosinase, and hemoglobin from bull blood. Comparison of the kinetic parameters of the oxidation reaction showed that peroxidase appeared to be the most effective biocatalyst in these processes. The specificity of the enzyme for quercetin increased with increasing the polarity of the solvent in a series of ethanolacetonitriledimethyl sulfoxide.
Asunto(s)
Catequina/química , Hemoglobinas/química , Peroxidasa de Rábano Silvestre/química , Monofenol Monooxigenasa/química , Quercetina/análogos & derivados , Quercetina/química , Acetonitrilos/química , Agaricales/química , Agaricales/enzimología , Animales , Bovinos , Dimetilsulfóxido/química , Etanol/química , Proteínas Fúngicas/química , Cinética , Oxidación-Reducción , Soluciones , Solventes/químicaRESUMEN
We present the structure of the decanucleotide d(CGTATATACG) determined by single crystal X-ray diffraction at 1.58 A resolution. A netropsin drug is found in the minor groove with guanine stacked on a pyrrole ring of the drug, a feature described here for the first time. The stacked guanine is an extra-helical base coming from the end of a neighbour oligonucleotide. This observation may open the way to the development of minor groove binding drugs with a higher sequence selectivity. The oligonucleotide is in the B-conformation, but the terminal base-pairs are disrupted: the cytosine residues are disordered while the guanine residues penetrate into the minor groove of neighbouring duplexes. Four hydrated Ni ions with octahedral co-ordination are found associated with the N7 atoms of each guanine. The high affinity of these ions with guanine suggests that they may be used as probes for specific guanine residues.
Asunto(s)
Guanina/metabolismo , Netropsina/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Níquel/metabolismo , Relación Estructura-ActividadRESUMEN
We have determined the structure of the dodecamer duplex d(CGCCCGCGGGCG)2. A careful use of the molecular replacement programme AMoRe has been essential in order to solve the structure. This dodecamer shows a unique conformation, quite different from all the previously studied oligonucleotide duplexes: the central octamer has an A conformation, but with a sharp 65 degrees kink in the centre; the terminal base-steps have a B-like conformation; the major groove is completely closed in the centre, a hollow molecule is thus found. The results obtained confirm the high degree of variability of DNA structure. A new type of kink and an intermediate A/B double-helical conformation have been found. Such intermediate conformation differs from those described in DNA polymerase complexes.
Asunto(s)
ADN/química , Oligodesoxirribonucleótidos/química , Emparejamiento Base , Secuencia de Bases , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido NucleicoRESUMEN
We have solved the single crystal X-ray structure of the synthetic DNA hexamer d(CCGCGG). The central alternating tetramer forms a Z-DNA duplex. The initial cytosine of each strand of the duplex swings out and forms a Watson-Crick base-pair with the terminal guanine of a symmetry-related molecule. Thus, two symmetry-related DNA molecules form a twin with intermolecular base-pairs at both ends. Such a twin is additionally stabilized by a sodium ion located on a dyad axis between two DNA duplexes. The total structure has recombination-like features. It also provides a model for B/Z junctions. The crystal used in this study belongs to space group C222(1) with a = 34.33 A, b = 44.04 A and c = 38.27 A. The structure was solved by molecular replacement using partial models, and refined by molecular dynamics simulated annealing and positional treatment. The refinement has been concluded with an R-factor of 18.5% for 2377 reflections with F > or = 2 sigma (F) in the resolution region 8.0 to 1.92 A. The asymmetric unit contains two strands of d(CCGCGG) and 38 water molecules.
Asunto(s)
Modelos Moleculares , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Cristalización , Cristalografía por Rayos X , ADN/química , Fosfatos de Dinucleósidos/química , Estructura Molecular , Oligodesoxirribonucleótidos/síntesis química , Recombinación GenéticaRESUMEN
A new molecular mechanism of trinucleotide expansion diseases is suggested. The mechanism involves the formation of double-helical RNA hairpins by transcripts carrying (CNG)(n) sequences, which are processed via the RNAi pathway with subsequent RNA silencing of genes containing (CNG)(n) sequences. Depletion of proteins encoded by these genes leads to the specific disease phenotype. The available data on human myotonic dystrophy 1, which results from the (CTG)(n) expansion, support the hypothesis.
Asunto(s)
Interferencia de ARN , Expansión de Repetición de Trinucleótido , Secuencia de Bases , Silenciador del Gen , Trastornos Heredodegenerativos del Sistema Nervioso , Humanos , Conformación de Ácido Nucleico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
The title oligonucleotide and many related dodecamers have been extensively studied alone and as DNA-drug complexes. In practically all cases they were found to crystallize in the same space group, stabilized by interactions among the terminal guanine bases. Here we report new packing interactions (R3) in the presence of Ca2+. The oligonucleotides interact by placing their terminal guanines in the narrow groove of a neighbor molecule, an interaction which had never been found in dodecamers.
Asunto(s)
ADN/química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Sitios de Unión , Calcio , Cristalografía por Rayos X , Guanina , Modelos Moleculares , Preparaciones FarmacéuticasRESUMEN
A simplified multidimensional search was applied to determine the structure of deoxyhexamer CGCGCG in the crystal form belonging to the space group C222(1) (a = 45.6 A, b = 37.3 A, c = 70.3 A). This crystal form contains two Z-DNA duplexes per asymmetric unit in a similar orientation. The search consists of several main steps. As a first step, the analysis of packing modes is carried out and constraints for the position of duplexes in asymmetric unit are formulated. In order to choose true packing mode duplexes are represented as cylinders of a constant density and global search is carried out. Only reflections belonging to the plane in reciprocal space perpendicular to DNA axis were used in the calculations. An analytical representation of diffraction from a hollow cylinder is given which allows further refinement of corresponding parameters. During the global search the value of a linear correlation coefficient is estimated for each solution to pick up the best one. At the second step the "more local" search is carried out. The DNA model of a certain conformation (in our case Z-DNA) is taken and several parameters are being varied. These are: a) rotations of the independent duplexes around their axes (only two rotational parameters for two duplexes in our example) and b) a few positional parameters for which there is no corresponding constraint in the packing mode tested. In our case we had to vary only x1 and y2, where subindex corresponds to molecule number. The last stage concerned is quite similar to the multidimensional search realised in program ULTIMA. The only difference that the number of variable parameters is less in our search. This number for the case under consideration was 4 instead of 12 that would be necessary in a conventional multidimensional search.
Asunto(s)
Simulación por Computador , ADN/química , Modelos Moleculares , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
DNA location in the crystal of the nonspecific lambda cro/(GT)4.(AC)4 complex has been studied by the isomorphous replacement method using iodinated and brominated oligonucleotides. The results of the search for heavy atom positions combined with previously obtained molecular replacement data suggest that the DNA octamer occupies two overlapping positions, each of the two duplexes (GTGTGTGT).(ACACACAC) belonging to the same imaginary longer double helix and differing only in the shift by two base pairs along the common sugar-phosphate backbone. In the crystals of the heavy atom derivatives different orientations of the DNA octamer are observed as well. It seems reasonable that the DNA mobility of both kinds might be a common feature of crystals of nonspecific repressor/DNA complexes.
Asunto(s)
Proteínas de Unión al ADN , ADN/química , Proteínas Represoras/química , Secuencia de Bases , Cristalografía por Rayos X , ADN/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Represoras/metabolismo , Proteínas Virales , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Phase diagrams for several oligonucleotide duplex-spermine systems have been constructed. These diagrams characterize the duplex and spermine concentrations ranges in which crystalline precipitates are formed. All of them are wedge-like form. The slope of the upper branch of the diagram is determined by the oligonucleotide length. The position of the lower branch depends on both the nucleotide sequence and its length. The position of the lower branch depends on both the nucleotide sequence and its length. It has been shown that the addition to the system of MgCl2 and NaCl salts and MPD results in specific changes in the diagrams. A model for oligonucleotide duplex-spermine system has been suggested which explains the main characteristic features of the obtained phase diagrams. The experimental phase diagrams for the (pGpT)n (pApC)n-spermine system (n = 2,3,4) have been analyzed ion terms of this model and the values of the binding constants of spermine and Mg2+ ions binding to duplexes have been determined. It permitted to identify the complexes that precipitated in different regions of the phase diagrams under various conditions. The diagram obtained in the presence of a cobalt hexammine counterion is also considered. It has been shown that this phase diagram, in general, is similar to those obtained for the oligonucleotide duplex-spermine system.
Asunto(s)
ADN/efectos de los fármacos , Espermina/farmacología , Fenómenos Químicos , Precipitación Química , Química Física , Cristalización , ADN/metabolismo , ADN/ultraestructura , Glicoles/farmacología , Cloruro de Magnesio/farmacología , Modelos Químicos , Oligodesoxirribonucleótidos/metabolismo , Cloruro de Sodio/farmacología , Espermina/metabolismoRESUMEN
We have determined the structure of the synthetic DNA octamer d(pCpCpCpGpCpGpGpG) in five different crystal forms by single crystal X-ray diffraction. One crystal belongs to the space group P4(3)2(1)2 with a = b = 41.77, c = 25.15 A, whereas all others have the space group P2(1)2(1)2(1) with progressively decreasing unit cell volumes. In all crystals the octamer forms duplexes of A-DNA and all crystals display a similar packing mode, typical for A-DNA. The structure of the duplex varies from loose to very compact when going from one crystal form to another. The most compact form exhibits a volume of 995 A3 per base pair. Such a high density has never been found in A-DNA, being more characteristic of Z-DNA crystals. A comparison of the most with the least compact forms gives a RMS value of 1.7 A, with the distance between the phosphate centers through the major groove being almost twice shorter in the compact form. The phosphate-phosphate separation across the major groove in the compact form is extremely small, 0.7 A. The helical parameters also vary significantly in the various crystal forms. Differences in the helical twist can reach 13 degrees in the same step of the octamer in different crystal forms. The results prove that A-DNA is structurally very variable and demonstrate that the local structure of the same DNA fragment can strongly depend on the crystal environment.
Asunto(s)
Fragmentación del ADN , ADN/química , Variación Genética , Oligonucleótidos/química , Composición de Base , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Conformación de Ácido Nucleico , Programas InformáticosRESUMEN
Binding specificity of mutant V55C disulfide bonded dimeric lambda-Cro protein (CroVC) to double-stranded DNA (dsDNA) was studied using generic hexamer oligonucleotide microchip. The curves of dissociation of hybridized DNA in the presence and absence of CroVC were converted into the effective discriminant constants to assess the relevant thermodynamic equilibrium binding constants for dsDNA-protein complexes. Then, tiling of longer oligonucleotides with shorter oligomers was used to search for sequence motifs with the highest binding specificity similarly to sequencing by hybridization. The comparison of the deduced sequences with the known natural operator half-sites demonstrated the principal ability to discern and reconstruct the major parts of 7-mer motifs corresponding to the strongest binding of CroVC subunits. Our results show the applicability of generic microchips to the analysis of binding specificity in the case of multi-subunit DNA-binding proteins.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos , Bacteriófago lambda/metabolismo , Secuencia de Bases , ADN/química , Disulfuros , Datos de Secuencia Molecular , Mutación , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Unión Proteica , Espectrometría de Fluorescencia , Temperatura , Termodinámica , Factores de TiempoRESUMEN
Oligonucleotide crystallization technique based on the method of phase diagrams is described in detail with (pGpT)3.(pApC)3 hexamer as an example. The key point of the technique consists of dividing the multiparameter crystallization space into a set of regions, each of which corresponds to the precipitation of a duplex in complex with a certain number of counterions.
Asunto(s)
Cristalización , Ácidos Nucleicos Heterodúplex , Oligonucleótidos , Conformación de Ácido NucleicoRESUMEN
A formaldehyde-produced fixation of defects caused by a despiralizing action of a protein was studied in the case of DNA-RNAase A complex. The concentration of the defects fixed was measured by kinetic formaldehyde method (KF-method). It was shown that following processes take place in the complex in the presence of formaldehyde: (a) fixation of defects; (b) unwinding of DNA; (c) inactivation of the protein. The rates of all these processes depend on the concentration of formaldehyde, phi. At formaldehyde concentrations above some critical value phic the protein is inactivated before the defects are fixed. At phi less than phic the protein inactivation proceeds more slowly than the fixation of defects; at sufficiently low formaldehyde concentration no inactivation of protein occurs practically during the fixation time (20 min). The number of new defects formed during the time of fixation is linear with the formaldehyde concentration in the region where no inactivation of the protein occurs. Therefore the initial concentration of defects can be determined through an extrapolation to zero concentration of formaldehyde. On the basis of the data obtained a method is proposed for the evaluation of the number of defects in DNA caused by the despiralizing action of proteins. A model is proposed describing the behaviour of the complexes of DNA with despiralizing proteins in the presence of formaldehyde.
Asunto(s)
ADN , Endonucleasas , Ribonucleasas , Formaldehído , Calor , Cinética , Matemática , Unión Proteica , Desnaturalización ProteicaRESUMEN
It has been shown that formaldehyde has no marked physical effect upon DNA resulting in lowering of its melting temperature. The effect of lowering of DNA melting temperature observed earlier by other authors resulted from the process of unwinding of DNA due to chemical reactions of formaldehyde with reactive base groups.
Asunto(s)
ADN , Formaldehído , Fenómenos Químicos , Química , Cinética , Desnaturalización de Ácido Nucleico , TemperaturaRESUMEN
Even a small amount of formaldehyde is shown to induce a drop in the RNase A enzymatic activity. This drop is rapid from the start and then begins to be slower. A supposition was made on nature of the enzyme activity. Comparison of the effects of formaldehyde on the enzymatic and the destabilizing activity of RNase A was made. The effect of formaldehyde on the enzymatic activity does not correlate with its effect on the ability of RNase to destabilize the DNA double helix.
Asunto(s)
Formaldehído , Ribonucleasas , Catálisis , Fenómenos Químicos , Química , ADNRESUMEN
The DNA-cro-repressor complex crystals have been obtained, five DNA fragments of the same nucleotide sequence and different length being used. The rotation function for crystals of complexes with hexamer (pGpT)3 . (pApC)3 and with octamer (pGpT)3 . (pApC)3 have been calculated. The order of cro-DNA complex crystals is shown to vary with DNA length, the crystal of the complex with octamer being the most perfect among all investigated complexes.
Asunto(s)
Proteínas de Unión al ADN , ADN , Proteínas Represoras , Factores de Transcripción , Secuencia de Bases , Cristalización , Proteínas Virales , Proteínas Reguladoras y Accesorias Virales , Difracción de Rayos XRESUMEN
A set of experimental phase diagrams revealing the region of existence of microcrystals in mixture "(pGpT)n.(pApC)n+spermine", n = 2,3,4, was obtained. All diagrams are wedge-like with the slope of the upper branch and the level of the lower one depending on the oligonucleotidd length. The presence of MPD, MgCl2 and NaCl changes the form of the diagrams in a different manner. A model explaining the peculiar features of the diagrams for mixture "oligonucleotide duplex+spermine" is proposed. The analysis of the diagrams was carried out on the basis of this model and the values of the binding constants for binding of spermine and Mg2+ to duplexes were estimated. Some conclusions about the types of complexes, which may form microcrystals in different regions of diagrams were made.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Polirribonucleótidos , Espermina , Sitios de Unión , Cristalización , Glicoles/farmacología , Sustancias Macromoleculares , Magnesio/farmacología , Cloruro de Magnesio , Modelos Químicos , Cloruro de Sodio/farmacologíaRESUMEN
Two crystal forms of the self-complementary tetramer GpGpCpC have been obtained by phase diagram technique: P6(2)22/P6(4)22. a = b = 67.7 A, c = 105.6 A and P3(2)12/P3(1)12, a = b = 116.9 A. c = 116.4 A. Both crystals form diffract at least up to 3.2 A. Diffraction patterns of both crystal forms have strongest base-stacking reflections corresponding to the Bragg spacing 3.38 A which is typical for B-DNA. Moreover the self-rotation function of the first crystal form shows regular located two-fold pseudo-axes periodicity of which also indicates that this is B-conformation. The same conclusion can be reached on the basis of the crystal packing of the duplexes in the unit cell. It should be emphasized that this is a new example of B-DNA crystal packing.
Asunto(s)
ADN/química , Cristalización , Conformación de Ácido Nucleico , Difracción de Rayos XRESUMEN
A novel promising approach to the improvement of analytical properties of horseradish peroxidase based on its inclusion into self-assembled structures of chitosan is discussed. It is shown that the reasonable choice of a polyelectrolyte, a detailed investigation of its interaction with the enzyme and the conditions of the {peroxidase-polyelectrolyte} complex formation allow for stabilizing the biocatalyst in aqueous and aqueous-organic media without a substantial loss in its activity and developing corresponding analytical procedures and biosensors. The latter provides highly selective determination of a number of organic compounds and sensitive determination of heavy metal ions that becomes possible due to the specific interactions of the analytes with the polymer matrix. Besides, the application of the proposed analytical systems and biosensors provides the expansion of the range of the compounds, and poorly water soluble and slowly oxidized substrates of peroxidase as well, which could be determined and real samples which could be analyzed by enzymatic methods. Analytical performance of the developed spectrophotometric indicator procedures and biosensors based on the self-assembled complex {peroxidase-chitosan} is demonstrated in the determination of metal ions (Hg(II), Cd(II), and Pb(II)), phenothiazines (promazine, chloropromazine, and trifluoroperazine), phenolic compounds (phenol, hydroquinone, catechol, pyrogallol, quercetin, rutin, and esculetin), organic peroxides (tert-butyl peroxide, 2-butanone peroxide, and benzoyl peroxide) in various samples, including water-insoluble matrices.