RESUMEN
The temperature dependence of the partial heat capacity of the native protein structure in an aqueous solution has been analyzed. It is shown that the strictly linear temperature dependence is due to the contributions of the vibrational and conformational components, which indicates volume consistensy and the absence of conformational transitions up to the main two-state transition. The two-level structural and functional organization of the protein three-dimensional structure are considered in relation to the energy and conformational entropy properties in accordance with the principles of the organization of the protein macromolecule.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Calor , Proteínas , Termodinámica , Proteínas/química , Entropía , Temperatura , Desnaturalización ProteicaRESUMEN
In the course of sample preparation for Next Generation Sequencing (NGS), DNA is fragmented by various methods. Fragmentation shows a persistent bias with regard to the cleavage rates of various dinucleotides. With the exception of CpG dinucleotides the previously described biases were consistent with results of the DNA cleavage in solution. Here we computed cleavage rates of all dinucleotides including the methylated CpG and unmethylated CpG dinucleotides using data of the Whole Genome Sequencing datasets of the 1000 Genomes project. We found that the cleavage rate of CpG is significantly higher for the methylated CpG dinucleotides. Using this information, we developed a classifier for distinguishing cancer and healthy tissues based on their CpG islands statuses of the fragmentation. A simple Support Vector Machine classifier based on this algorithm shows an accuracy of 84%. The proposed method allows the detection of epigenetic markers purely based on mechanochemical DNA fragmentation, which can be detected by a simple analysis of the NGS sequencing data.
Asunto(s)
Metilación de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Línea Celular Tumoral , Islas de CpG , Fragmentación del ADN , Bases de Datos Genéticas , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Neoplasias/genética , Neoplasias/patología , Análisis de Secuencia de ADN , Máquina de Vectores de SoporteRESUMEN
The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the 'gas phase'. It was shown that specific values of the energy of weak intramolecular bonds (∆Hint), conformational free energy (∆Gconf) and entropy (∆Sconf) are the same for proteins with molecular weight 7-25 kDa. Using the energy value (∆Hint) and the proposed approach for estimation of the conformational entropy of native protein (SNC), numerical values of the absolute free energy (GNC) were obtained.
Asunto(s)
Conformación Proteica , Pliegue de Proteína , Proteínas/química , Termodinámica , Entropía , Temperatura , Agua/químicaRESUMEN
Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed "reads" are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.
RESUMEN
We performed thermodynamic analysis of temperature-induced unfolding of mesophilic and thermophilic proteins. It was shown that the variability in protein thermostability associated with pH-dependent unfolding or linked to the substitution of amino acid residues on the protein surface is evidence of the governing role of the entropy factor. Numerical values of conformational components in enthalpy, entropy and free energy which characterize protein unfolding in the "gas phase" were obtained. Based on the calculated absolute values of entropy and free energy, a model of protein unfolding is proposed in which the driving force is the conformational entropy of native protein, as an energy of the heat motion (T·S(NC)) increasing with temperature and acting as an factor devaluating the energy of intramolecular weak bonds in the transition state.
Asunto(s)
Desnaturalización Proteica , Pliegue de Proteína , Proteínas/química , Entropía , Calor , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , TermodinámicaRESUMEN
We investigated the phenomenon of ultrasonic cleavage of DNA by analyzing a large set of cleavage patterns of DNA restriction fragments using polyacrylamide gel electrophoresis. The cleavage intensity of individual phosphodiester bonds was found to depend on the nucleotide sequence and the position of the bond with respect to the ends of the fragment. The relative intensities of cleavage of the central phosphodiester bond in 16 dinucleotides and 256 tetranucleotides were determined by multivariate statistical analysis. We observed a remarkable enhancement of the mean values of the relative intensities of cleavage (cleavage rates) in phosphodiester bonds following deoxycytidine, which diminished in the row of dinucleotides: d(CpG) > d(CpA) > d(CpT) >> d(CpC). The cleavage rates for all pairs of complementary dinucleotides were significantly different from each other. The effect of flanking nucleotides in tetranucleotides on cleavage rates of all 16 types of central dinucleotides was also statistically significant. The sequence-dependent ultrasonic cleavage rates of dinucleotides are consistent with reported data on the intensity of the conformational motion of their 5'-deoxyribose. As a measure of local conformational dynamics, cleavage rates may be useful for characterizing functional regions of the genome.
Asunto(s)
ADN/genética , ADN/metabolismo , Ultrasonido/métodos , Secuencia de Bases , ADN/química , Electroforesis en Gel de Poliacrilamida , Fenómenos Físicos , Docilidad , SolucionesRESUMEN
Distributions of phosphate backbone-produced electrostatic potentials around several tRNAs were calculated by solving the nonlinear Poisson-Boltzmann equation. The tRNAs were either free or bound to the proteins involved in translation: aminoacyl-tRNA and elongation factor EF-Tu. We identified several regions of strong negative potential related to typical structural patterns of tRNA and invariant throughout the tRNAs. The patterns are conserved upon binding of tRNAs to the synthetase and the EF-Tu. Variation of tRNA charge in our theoretical calculations of electrostatic potential-mediated pK shifts of pH-dependent labels attached to tRNA, compared to experimentally observed pK shifts for those labels, shows that the total charge of tRNA is large, within the interval of -40 to -70 proton charges. The electrostatic field of tRNA is sufficient to cause ionization of histidine residues of ARSase, causing additional free energy of ARSase-tRNA interaction of at least several kcal/mol. This may discriminate proteins with respect to the particular tRNA at large distances. Two types of tRNA-protein electrostatic recognition mechanisms are discussed. One, more specific, involves charges induced on protein by the large electrostatic potential of tRNA, while the other, less specific, does not involve induced charges.