RESUMO
In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.
Assuntos
Receptor de Endotelina B/química , Receptor de Endotelina B/isolamento & purificação , Animais , Baculoviridae/genética , Compostos de Bifenilo/química , Western Blotting , Dipeptídeos/química , Antagonistas dos Receptores de Endotelina/química , Endotelinas/química , Engenharia Genética/métodos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/química , Desnaturação Proteica , Estabilidade Proteica , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , TemperaturaRESUMO
BACKGROUND: The closed circular, multinucleosome-bound DNA comprising a minichromosome provides one of the best known examples of chromatin organization beyond the wrapping of the double helix around the core of histone proteins. This higher level of chain folding is governed by the topology of the constituent nucleosomes and the spatial disposition of the intervening protein-free DNA linkers. RESULTS: By simplifying the protein-DNA assembly to an alternating sequence of virtual bonds, the organization of a string of nucleosomes on the minichromosome can be treated by analogy to conventional chemical depictions of macromolecular folding in terms of the bond lengths, valence angles, and torsions of the chain. If the nucleosomes are evenly spaced and the linkers are sufficiently short, regular minichromosome structures can be identified from analytical expressions that relate the lengths and angles formed by the virtual bonds spanning the nucleosome-linker repeating units to the pitch and radius of the organized quaternary structures that they produce. CONCLUSIONS: The resulting models with 4-24 bound nucleosomes illustrate how a minichromosome can adopt the low-writhe folding motifs deduced from biochemical studies, and account for published images of the 30 nm chromatin fiber and the simian virus 40 (SV40) nucleohistone core. The marked sensitivity of global folding to the degree of protein-DNA interactions and the assumed nucleosomal shape suggest potential mechanisms for chromosome rearrangements upon histone modification.
Assuntos
Cromossomos , Nucleossomos/química , Dobramento de Proteína , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/químicaRESUMO
A low-resolution molecular model, which combines the known mechanical properties of protein-free DNA with the accumulating picture of chromatosome structure, has been developed to account for the stretching of single chromatin fibers by an imposed external force. Force-extension characteristics of sets of chains accumulated by Monte Carlo sampling are consistent with recently observed findings in the non-destructive regime (<20 pN imposed force), where the structure of the chromatosome remains intact. The correspondence between simulation and the relaxation phase of the experiment limits the equilibrium entry-exit angle of linker DNA on the chromatosome to W=50(+/-10) degrees and the effective DNA linker length to L(eff)=40(+/-5) bp. The computed force-extension characteristics are relatively insensitive to other parameters of the model, precluding their accurate estimation. The introduction of an attractive potential between closely spaced nucleosomes reproduces the added initial resistance of single fibers to extension at high salt conditions. The consideration of elastic linkers also improves the fitting of assorted classical measurements of unstressed chromatin structure in solution. The overall picture of chromatin that emerges is an irregular, fluctuating, three-dimensional, zig-zag structure with intact, mechanically stable chromatosome units and deformable linkers. The modeled fiber undergoes large-scale configurational rearrangements without significant perturbation of the constituent chromatosome beads, collapsing into a highly condensed form in response to small (<2kT) inter-nucleosomal attractions.
Assuntos
Cromatina/química , Cromatina/metabolismo , Simulação por Computador , Calibragem , DNA/química , DNA/metabolismo , Elasticidade , Modelos Moleculares , Conformação Molecular , Método de Monte Carlo , Nucleossomos/química , Nucleossomos/metabolismo , Concentração Osmolar , Estimulação Física , Maleabilidade , TermodinâmicaRESUMO
Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.
Assuntos
Dano ao DNA , DNA/química , Cloreto de Magnésio/farmacologia , Conformação de Ácido Nucleico , Cloreto de Sódio/farmacologia , DNA/metabolismo , Congelamento , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Modelos Moleculares , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismoRESUMO
We use a combination of spectroscopic, calorimetric, viscometric and computer modeling techniques to characterize the binding of the aminoglycoside antibiotic, tobramycin, to the polymeric RNA duplex, poly(rI).poly(rC), which exhibits the characteristic A-type conformation that is conserved among natural and synthetic double-helical RNA sequences. Our results reveal the following significant features: (i) CD-detected binding of tobramycin to poly(rI).poly(rC) reveals an apparent site size of four base-pairs per bound drug molecule; (ii) tobramycin binding enhances the thermal stability of the host poly(rI).poly(rC) duplex, the extent of which decreases upon increasing in Na(+) concentration and/or pH conditions; (iii) the enthalpy of tobramycin- poly(rI).poly(rC) complexation increases with increasing pH conditions, an observation consistent with binding-induced protonation of one or more drug amino groups; (iv) the affinity of tobramycin for poly(rI).poly(rC) is sensitive to both pH and Na(+) concentration, with increases in pH and/or Na(+) concentration resulting in a concomitant reduction in binding affinity. The salt dependence of the tobramycin binding affinity reveals that the drug binds to the host RNA duplex as trication. (v) The thermodynamic driving force for tobramycin- poly(rI).poly(rC) complexation depends on pH conditions. Specifically, at pH< or =6.0, tobramycin binding is entropy driven, but is enthalpy driven at pH > 6.0. (vi) Viscometric data reveal non-intercalative binding properties when tobramycin complexes with poly(rI).poly(rC), consistent with a major groove-directed mode of binding. These data also are consistent with a binding-induced reduction in the apparent molecular length of the host RNA duplex. (vii) Computer modeling studies reveal a tobramycin-poly(rI). poly(rC) complex in which the drug fits snugly at the base of the RNA major groove and is stabilized, at least in part, by an array of hydrogen bonding interactions with both base and backbone atoms of the host RNA. These studies also demonstrate an inability of tobramycin to form a stable low-energy complex with the minor groove of the poly(rI).poly(rC) duplex. In the aggregate, our results suggest that tobramycin-RNA recognition is dictated and controlled by a broad range of factors that include electrostatic interactions, hydrogen bonding interactions, drug protonation reactions, and binding-induced alterations in the structure of the host RNA. These modulatory effects on tobramycin-RNA complexation are discussed in terms of their potential importance for the selective recognition of specific RNA structural motifs, such as asymmetric internal loops or hairpin loop-stem junctions, by aminoglycoside antibiotics and their derivatives.
Assuntos
Antibacterianos/metabolismo , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Tobramicina/metabolismo , Antibacterianos/química , Pareamento de Bases/efeitos dos fármacos , Pareamento de Bases/genética , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Simulação por Computador , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peso Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Prótons , Estabilidade de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , Sódio/farmacologia , Espectrofotometria Ultravioleta , Eletricidade Estática , Especificidade por Substrato , Termodinâmica , Titulometria , Tobramicina/química , ViscosidadeRESUMO
Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.
Assuntos
DNA/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , Sequência de Bases , Congelamento , Modelos Químicos , Dados de Sequência Molecular , Método de Monte CarloRESUMO
Various site-specific recombination enzymes produce different types of knots or catenanes while acting on circular DNA in vitro and in vivo. By analysing the types of knots or links produced, it is possible to reconstruct the order of events during the reaction and to deduce the molecular "architecture" of the complexes that different enzymes form with DNA. Until recently it was necessary to use laborious electron microscopy methods to identify the types of knots or catenanes that migrate in different bands on the agarose gels used to analyse the products of the reaction. We reported recently that electrophoretic migration of different knots and catenanes formed on the same size DNA molecules is simply related to the average crossing number of the ideal representations of the corresponding knots and catenanes. Here we explain this relation by demonstrating that the expected sedimentation coefficient of randomly fluctuating knotted or catenated DNA molecules in solution shows approximately linear correlation with the average crossing number of ideal configurations of the corresponding knots or catenanes.
Assuntos
DNA Circular/química , Eletroforese em Gel de Ágar , Conformação de Ácido Nucleico , Centrifugação , Modelos Lineares , Modelos MolecularesRESUMO
Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature.
Assuntos
DNA Circular/química , Modelos Teóricos , Conformação de Ácido Nucleico , Simulação por Computador , Modelos Estruturais , Método de Monte Carlo , TermodinâmicaRESUMO
Long polymers in solution frequently adopt knotted configurations. To understand the physical properties of knotted polymers, it is important to find out whether the knots formed at thermodynamic equilibrium are spread over the whole polymer chain or rather are localized as tight knots. We present here a method to analyze the knottedness of short linear portions of simulated random chains. Using this method, we observe that knot-determining domains are usually very tight, so that, for example, the preferred size of the trefoil-determining portions of knotted polymer chains corresponds to just seven freely jointed segments.
Assuntos
Conformação Molecular , Polímeros/química , Modelos Moleculares , Distribuição AleatóriaRESUMO
The shortest tube of constant diameter that can form a given knot represents the 'ideal' form of the knot. Ideal knots provide an irreducible representation of the knot, and they have some intriguing mathematical and physical features, including a direct correspondence with the time-averaged shapes of knotted DNA molecules in solution. Here we describe the properties of ideal forms of composite knots-knots obtained by the sequential tying of two or more independent knots (called factor knots) on the same string. We find that the writhe (related to the handedness of crossing points) of composite knots is the sum of that of the ideal forms of the factor knots. By comparing ideal composite knots with simulated configurations of knotted, thermally fluctuating DNA, we conclude that the additivity of writhe applies also to randomly distorted configurations of composite knots and their corresponding factor knots. We show that composite knots with several factor knots may possess distinct structural isomers that can be interconverted only by loosening the knot.
Assuntos
Simulação por Computador , Modelos Químicos , Conformação de Ácido Nucleico , Modelos MolecularesRESUMO
The concept of ideal geometric configurations was recently applied to the classification and characterization of various knots. Different knots in their ideal form (i.e., the one requiring the shortest length of a constant-diameter tube to form a given knot) were shown to have an overall compactness proportional to the time-averaged compactness of thermally agitated knotted polymers forming corresponding knots. This was useful for predicting the relative speed of electrophoretic migration of different DNA knots. Here we characterize the ideal geometric configurations of catenanes (called links by mathematicians), i.e., closed curves in space that are topologically linked to each other. We demonstrate that the ideal configurations of different catenanes show interrelations very similar to those observed in the ideal configurations of knots. By analyzing literature data on electrophoretic separations of the torus-type of DNA catenanes with increasing complexity, we observed that their electrophoretic migration is roughly proportional to the overall compactness of ideal representations of the corresponding catenanes. This correlation does not apply, however, to electrophoretic migration of certain replication intermediates, believed up to now to represent the simplest torus-type catenanes. We propose, therefore, that freshly replicated circular DNA molecules, in addition to forming regular catenanes, may also form hemicatenanes.