RESUMEN
Small monomeric proteins from mesophilic and thermophilic organisms were studied. They have close structural and physical and chemical properties but vary in thermal stability. A thermodynamic analysis of heat unfolding was made and integral enthalpy of unfolding (DeltaH(unf)), heat capacity of hydration (DeltaC(p)(hyd)) and enthalpy of hydration (DeltaH(hyd)) and of the buried surface area (DeltaASA) of nonpolar and polar groups as well as the enthalpy of disruption of intramolecular interaction (DeltaH(int) in gas phase) at 298 K were determined. The absence of correlation between protein thermostability and energetic components suggests that regulatory mechanism of protein thermal stabilization has entropic nature.
Asunto(s)
Metabolismo Energético , Pliegue de Proteína , Proteínas/química , Proteínas/metabolismo , Desecación , Enlace de Hidrógeno , Modelos Químicos , Desnaturalización Proteica , Temperatura , TermodinámicaRESUMEN
The conformational stability of the rat thyroid transcription factor 1 homeodomain, TTF-1HD, has been investigated by means of circular dichroism (CD) and differential scanning calorimetry (DSC) measurements at pH 5.0 as a function of KCl concentration. Thermal unfolding of TTF-1HD is a reversible two-state transition. The protein is not stable against temperature, showing a denaturation temperature of 32 degrees C in the absence of salt and 50 degrees C at 75 mM KCl. The binding energetics of TTF-1HD to its target DNA sequence has been characterized by means of isothermal titration calorimetry (ITC) measurements, complemented with CD data. At 25 degrees C, pH 5.0 and 75 mM KCl, the binding constant amounts to 1.5 x 10(8)M(-1) and the binding enthalpy change amounts to -41 kJ mol(-1). The process is enthalpy driven, but also the entropy change is favorable to complex formation. To gain a molecular level understanding of the interactions determining the association of TTF-1HD to the target DNA sequence structural information would be requested, but it is not yet available. Therefore, structural models of two complexes, TTF-1HD with the target DNA sequence and TTF-1HD with a modified DNA sequence, have been constructed by using as a template the NMR structure of the complex between NK-2 HD and its target DNA, and by performing molecular dynamics simulations 3.5 ns long. Analysis of these models allows one to shed light on the origin of the DNA binding specificity characteristic of TTF-1HD.
Asunto(s)
ADN/química , Proteínas de Homeodominio/química , Proteínas Nucleares/química , Termodinámica , Factores de Transcripción/química , Animales , Sitios de Unión , Calorimetría , Dicroismo Circular , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformación Proteica , Ratas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/metabolismoRESUMEN
In order to clarify the role played by the N-terminal region for the conformational stability of the thermophilic esterase 2 (EST2) from Alicyclobacillus acidocaldarius, two mutant forms have been investigated: a variant obtained by deleting the first 35 residues at the N-terminus (EST2-36del), and a variant obtained by mutating Lys102 to Gln (K102Q) to perturb the N-terminus by destroying the salt bridge E43-K102. The temperature- and denaturant-induced unfolding of EST2 and the two mutant forms have been studied by means of circular dichroism (CD), differential scanning calorimetry (DSC) and fluorescence measurements. In line with its thermophilic origin, the denaturation temperature of EST2 is high: T(d)=91 degrees C and 86 degrees C if detected by recording the CD signal at 222 nm and 290 nm, respectively. This difference suggests that the thermal denaturation process, even though reversible, is more complex than a two-state Nright arrow over left arrowD transition. The non-two-state behaviour is more pronounced in the case of the two mutant forms. The complex DSC profiles of EST2 and both mutant forms have been analysed by means of a deconvolution procedure. The thermodynamic parameters characterizing the two transitions obtained in the case of EST2 are: T(d,1)=81 degrees C, Delta(d)H(1)=440 kJ mol(-1), Delta(d)C(p,1)=7 kJ K(-1)mol(-1), T(d,2)=86 degrees C, Delta(d)H(2)=710 kJ mol(-1), and Delta(d)C(p,2)=9 kJ K(-1)mol(-1). The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 degrees C. The results indicate that EST2 possesses two structural domains whose coupling is tight in the wild-type protein, but markedly weakens in the two mutant forms as a consequence of the perturbations in the N-terminal region.
Asunto(s)
Proteínas Bacterianas/química , Esterasas/química , Bacilos Grampositivos Formadores de Endosporas/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Estabilidad de Enzimas , Esterasas/genética , Esterasas/aislamiento & purificación , Mutación , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H2TMPyP to the two DNA quadruplexes, [d(AGGGT)]4 and [d(TGGGGT)]. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]4 and 2:1 for [d(TGGGGT)], respectively.
Asunto(s)
Ácidos Nucleicos Heterodúplex , Porfirinas/química , Fenómenos Biofísicos , Biofisica , Calorimetría , ADN/química , Relación Dosis-Respuesta a Droga , Cinética , Ligandos , Modelos Químicos , Conformación Molecular , Conformación de Ácido Nucleico , Temperatura , Termodinámica , Factores de TiempoRESUMEN
The aim of this work is to compare the physicochemical properties of three oligonucleotidic sequences, d(TGGGT), d(TGGGGT) and d(TGGGGGT), which assemble to form quadruplex structures with the same molecularity, but containing three, four, and five G-quartets, respectively. The addition of one or two G-tetrads greatly increases both the enthalpy and Tm values of the quadruplex dissociation.
Asunto(s)
Biología Molecular/métodos , Ácidos Nucleicos Heterodúplex , Fenómenos Biofísicos , Biofisica , Dicroismo Circular , ADN/química , Electroforesis en Gel de Poliacrilamida , Calor , Conformación Molecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , Temperatura , TermodinámicaRESUMEN
The effects of cytosine protonation on the thermodynamic properties of parallel pyrimidine motif DNA triplex were investigated and characterized by different techniques, such as circular dichroism (CD), ultraviolet spectroscopy (UV) and differential scanning calorimetry (DSC). A thermodynamic model was developed which, by linking the cytosine ionization equilibrium to the dissociation process of the triplex, is able to rationalize the experimental data and to reproduce the pH dependence of the free energy, enthalpy and entropy changes associated with the triplex formation. The results are useful to systematically introduce the effect of pH in a more general model able to predict the stability of DNA triplexes on the basis of the sequence alone.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Protones , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Citosina/química , Concentración de Iones de Hidrógeno , Espectrofotometría Ultravioleta , Temperatura , TermodinámicaRESUMEN
The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.
Asunto(s)
Dicroismo Circular , Esterasas/química , Trifluoroetanol/química , Animales , Estabilidad de Enzimas , Conformación Proteica , Temperatura , TermodinámicaRESUMEN
The unfolding induced by guanidine hydrochloride of the small protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus has been investigated by means of circular dichroism and fluorescence measurements. At neutral pH and room temperature the midpoint of the transition occurred at 4M guanidine hydrochloride. Thermodynamic information was obtained by means of both the linear extrapolation model and the denaturant binding model, in the assumption of a two-state N<==>D transition. A comparison with thermodynamic data determined from the thermal unfolding of Sso7d indicated that the denaturant binding model has to be preferred. Finally, it is shown that Sso7d is the most stable against both temperature and guanidine hydrochloride among a set of globular proteins possessing a very similar 3D structure.
Asunto(s)
Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Guanidina/química , Sulfolobus solfataricus/química , Dicroismo Circular , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Telomeric guanine-rich sequence can adopt quadruplex structures that are important for their biological role in chromosomal stabilisation. G quartets are characterised by the cyclic hydrogen bonding of four guanine bases in a coplanar arrangement and their stability is ion-dependent. In this work we compare the stability of [d(TGGGT)](4) and [d(T*GGGT)](4) quadruplexes. The last one contains a modified thymine, where the hydroxyl group substitutes one hydrogen atom of the methyl group of the thymine in the [d(TGGGT)](4) sequence. We used a combination of spectroscopic, calorimetric and computational techniques to characterise the G-quadruplex formation. NMR and CD spectra of [d(T*GGGT)](4) were characteristic of parallel-stranded, tetramolecular quadruplex. CD and DSC melting experiments reveal that [d(T*GGGT)](4) is less stable that unmodified quadruplex. Molecular models suggest possible explanation for the observed behaviour.
Asunto(s)
ADN/química , Timina/química , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Guanina/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Temperatura , Factores de TiempoRESUMEN
A NMR structural study of quadruplex [d(TGGGT)]4 containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.
Asunto(s)
Guanina/química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis químicaRESUMEN
Starting from the discovery, in the mid-1930s, that petroleum pipelines in the colder regions of the Northern hemisphere contained crusts of some crystals, and were often blocked by them, a short history of the development of research on the structures, properties, and possible exploitation of the class of inclusion compounds known as gas hydrates is given. The state of the assessment of the natural reservoirs and their perspectives for exploitation are presented, together with an analysis of the hypotheses on the origins of the hydrates. Finally, the phase diagrams are shown in relation to environmental problems arising from the instability of the hydrate fields due to global warming or geological activity.
Asunto(s)
Combustibles Fósiles , Agua/química , Combustibles Fósiles/toxicidad , Calentamiento Global , InternacionalidadRESUMEN
In the last years we have performed a series of studies to characterize the conformational stability of three esterases from thermophilic and mesophilic sources: Aes esterase from Escherichia coli, EST2 from Alicyclobacillus acidocaldarius and AFEST from Archeoglobus fulgidus. These three esterases belong to the Hormone-sensitive lipase group of the superfamily of carboxylester hydrolases. The conformational stability of the three enzymes against temperature, urea and GuHCl has been determined by means of circular dichroism, fluorescence and differential scanning calorimetry measurements. Analysis of experimental data coupled with available structural information allowed us to suggest that the optimization of charge-charge interactions on the protein surface could one of the mechanisms to increase the thermal stability for the three esterases. This idea has been tested in the case of EST2, which shows a fully reversible thermal unfolding, by producing and studying variant forms of wild type enzyme in which a charged residue has been mutated. In the present article the obtained results are critically recollected in order to provide a clear and unified scenario.
Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Esterasas/química , Conformación Proteica , Alicyclobacillus/enzimología , Alicyclobacillus/genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Archaeoglobales/enzimología , Archaeoglobales/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Esterasas/genética , Esterasas/metabolismo , Guanidina/química , Cinética , Mutación , Pliegue de Proteína , Estructura Terciaria de Proteína , Esterol Esterasa/química , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Temperatura , Urea/químicaRESUMEN
Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. The guanine rich strand can fold into four-stranded G-quadruplex structures, which have recently become attractive for biomedical research. Indeed, the aptamers based on the quadruplex motif may prove useful as tools aimed at binding and inhibiting particular proteins, catalyzing various biochemical reactions, or even serving as pharmaceutically active agents. The incorporation of modified bases into oligonucleotides can have profound effects on their folding and may produce useful changes in physical and biological properties of the resulting DNA fragments. In this work, the adenines of the human telomeric repeat oligonucleotide d(TAGGGT) and d(AGGGT) were substituted by 2'-deoxy-8-(propyn-1-yl)adenosine (A-->APr) or by 8-bromodeoxyadenosine (A-->ABr). The biophysical properties of the resulting quadruplex structures were compared with the unmodified quadruplexes. NMR and CD spectra of the studied sequences were characteristic of parallel-stranded, tetramolecular quadruplexes. The analysis of the equilibrium melting curves reveals that the modifications stabilize the quadruplex structure. The results are useful when considering the design of novel aptameric nucleic acids with diverse molecular recognition capabilities that would not be present using native RNA/DNA sequences.
Asunto(s)
ADN/química , Desoxiadenosinas/química , Telómero/química , Dicroismo Circular , G-Cuádruplex , Humanos , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Secuencias Repetidas en Tándem/genética , Telómero/genética , Termodinámica , Temperatura de TransiciónRESUMEN
Rat brain Fe65 and its truncated forms corresponding to the combined PTB1 and PTB2 domains, as well as to the isolated PTB2 domain, were expressed in Escherichia coli and purified from inclusion bodies by affinity chromatography. The recombinant proteins were refolded and judged functionally active by their ability to interact with native APP. Limited proteolysis of recombinant Fe65 and PTB1-2 with trypsin, chymotrypsin and V8 proteases showed that the most sensitive proteoltytic sites were positioned at the level of the interdomain regions comprised between WW/PTB1 and PTB1/PTB2. Secondary structure of the recombinant proteins, evaluated by CD spectroscopy, showed a different degree of unordered structures, the PTB2 domain being the higher organised region. In addition, intrinsic fluorescence measurements of PTB2, indicated that a conformational transition of the protein can be induced by denaturating agents such as GuHCl. These data provide first evidences on the secondary structural levels of Fe65.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Fosfotirosina/metabolismo , Animales , Sitios de Unión , Western Blotting , Química Encefálica/fisiología , Dicroismo Circular , Expresión Génica , Hidrólisis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plásmidos/genética , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to complementary single-strand (DNA or RNA), and the resistance to nuclease and protease degradation. At the same time, the limitations of an oligomer containing all PNA residues, such as low water solubility, self-aggregation, and low cellular uptake, are effectively overcome. Further, PNA-DNA chimeras possess interesting biological properties as antisense agents. We have explored the ability of PNA-DNA chimeric strands to assemble in quadruplex structures. The rate constant for association of the quadruplexes and their thermodynamic properties have been determined by CD spectroscopy and differential scanning calorimetry (DSC). Thermal denaturation experiments indicated higher thermal and thermodynamic stabilities for chimeric quadruplexes in comparison with the corresponding unmodified DNA quadruplex. Singular value decomposition analysis (SVD) suggests the presence of kinetically stable intermediate species in the quadruplex formation process. The experimental results have been discussed on the basis of molecular dynamic simulations. The ability of PNA-DNA chimeras to form stable quadruplex structures expands their potential utility as therapeutic agents.
Asunto(s)
ADN/química , Ácidos Nucleicos de Péptidos/química , Calorimetría , Dicroismo Circular , G-Cuádruplex , Cinética , Modelos Químicos , Conformación de Ácido Nucleico , Conformación Proteica , Temperatura , TermodinámicaRESUMEN
Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation. Telomeric sequences can form quadruplex structures stabilized by guanine quartets. These structures can be constructed from one, two, or four oligonucleotidic strands. Here, we report the thermodynamic characterization of the stability, analyzed by differential scanning calorimetry, of three DNA quadruplexes of different molecularity, all containing four G-tetrads. The conformational properties of these quadruple helices were studied by circular dichroism. The investigated oligomers form well-defined G-quadruplex structures in the presence of sodium ions. Two have the truncated telomeric sequence from Oxytricha, d(TGGGGT) and d(GGGGTTTTGGGG), which form a tetramolecular and bimolecular quadruplex, respectively. The third sequence, d(GGGGTTGGGGTGTGGGGTTGGGG) was designed to form a unimolecular quadruplex. The thermodynamic parameters of these quadruplexes have been determined. The tetramolecular structure is thermodynamically more stable than the bimolecular one, which, in turn, is more stable than the unimolecular one. The experimental data were discussed in light of the molecular-modeling study.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Telómero/química , Secuencia de Bases , Rastreo Diferencial de Calorimetría , Dicroismo Circular , ADN/síntesis química , G-Cuádruplex , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Oligonucleótidos/síntesis química , Telómero/genética , TermodinámicaRESUMEN
The stability of acetyl-esterase, Aes, from Escherichia coli against the denaturing action of urea and guanidine hydrochloride, GuHCl, has been investigated by means of circular dichroism and fluorescence measurements. The urea-induced unfolding curves show a single inflection point at 6.2 M urea, whereas the GuHCl-induced curves show two inflection points at 1.4 and 3.1 M GuHCl. The unfolding process is reversible with both urea and GuHCl. These results, together with similar experimental data on the mutant form V20D-Aes, suggest the presence of two domains in the Aes structure, which unfold more or less independently depending on the denaturant used. This is also supported by a 3D model obtained by homology modeling using the structure of brefeldine as a template. The effect of NaCl on the urea-induced unfolding curves of the enzyme has also been investigated.
Asunto(s)
Acetilesterasa/química , Proteínas de Escherichia coli/química , Guanidina , Pliegue de Proteína , Urea , Acetilesterasa/genética , Ácido Aspártico/genética , Hidrolasas de Éster Carboxílico/química , Dicroismo Circular , Estabilidad de Enzimas/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutación Puntual , Desnaturalización Proteica , Estructura Terciaria de Proteína/genética , Cloruro de Sodio , Espectrometría de Fluorescencia , Homología Estructural de Proteína , Termodinámica , Valina/genéticaRESUMEN
Sso7d is a 62-residue, basic protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Around neutral pH, it exhibits a denaturation temperature close to 100 degrees C and a non-sequence-specific DNA binding activity. Here, we report the characterization by circular dichroism and fluorescence measurements of a variant form of Sso7d truncated at leucine 54 (L54Delta). It is shown that L54Delta has a folded conformation at neutral pH and that its thermal unfolding is a reversible process, represented well by the two-state N <=> D transition model, with a denaturation temperature of 53 degrees C. Fluorescence titration experiments indicate that L54Delta binds tightly to calf thymus DNA, even though the binding parameters are smaller than those of the wild-type protein. Therefore, the truncation of eight residues at the C-terminus of Sso7d markedly affects the thermal stability of the protein, which nevertheless retains a folded structure and DNA binding activity.
Asunto(s)
Proteínas Arqueales , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/metabolismo , Leucina/química , Sulfolobus/metabolismo , Dicroismo Circular , Proteínas de Unión al ADN/química , Fluorescencia , Calor , Sulfolobus/químicaRESUMEN
We studied the temperature- and denaturant-induced denaturation of two thermophilic esterases, AFEST from Archeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, by means of circular dichroism measurements. Both enzymes showed a very high denaturation temperature: 99 degrees C for AFEST and 91 degrees C for EST2. They also showed a remarkable resistance against urea; at half-completion of the transition the urea concentration was 7.1 M for AFEST and 5.9 M for EST2. On the contrary, both enzymes showed a weak resistance against GuHCl; at half-completion of the transition the GuHCl concentration was 2.0 M for AFEST and 1.9 M for EST2. The thermodynamic parameters characterizing urea- and GuHCl-induced denaturation of the studied enzymes have been obtained by both the linear extrapolation model and the denaturant binding model. The dependence of the thermal stability on NaCl concentration for both esterases has also been determined. A careful analysis of the data, coupled with available structural information, has allowed the proposal of a reliable interpretation.
Asunto(s)
Esterasas/química , Desnaturalización Proteica , Bacillus/enzimología , Dicroismo Circular , Estabilidad de Enzimas , Guanidina/química , Conformación Proteica , Pliegue de Proteína , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Urea/químicaRESUMEN
The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for the stability of the two esterases.