RESUMO
Malate dehydrogenase from Haloarcula marisomortui (hMDH) is active, soluble and mildly unstable in an unusually wide range of salt conditions and temperatures, making it a particularly interesting model for the study of solvent effects on protein stability. Its denaturation (loss of activity due to concomitant dissociation and unfolding) kinetics was studied as a function of temperature and concentration of NaCl, potassium phosphate or ammonium sulphate in H2O or 2H2O. A transition-state-theory analysis was applied to the data. In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when 2H2O replaced H2O. Each salt condition was associated with a particular energy regime that dominated stability. In NaCl/H2O, a positive enthalpy term, delta H not equal to 0, always dominated the activation free energy of denaturation, delta G not equal to 0. In potassium phosphate/H2O and ammonium sulphate/H2O, on the other hand, stability was dominated by a negative activation entropy, delta S not equal to 0. and delta H not equal to 0 changed sign between 10 degrees C and 20 degrees C, consistent with a strong hydrophobic effect contribution, in these salting-out solvents. Decreasing stability at low temperatures, favouring cold denaturation, was observed. Replacing H2O by 2H2O strengthened the hydrophobic effect in all conditions. As a consequence, conditions were found in which hMDH was not halophilic; below 10 degrees C, it was stable in approximately 0.1 M NaCl/2H2O. The solution structure and preferential solvent interactions of hMDH in H2O or 2H2O solvents containing NaCl were studied by densimetry and neutron scattering. Despite the different stability of the protein in H2O or 2H2O, an experimentally identical invariant solution particle was formed in both solvents. It had a total volume of 1.165 cm3 g-1 and bound about 0.4 g of H2O (0.44 g of 2H2O) and about 0.08 g NaCl g protein. The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.
Assuntos
Malato Desidrogenase/química , Desnaturação Proteica , Sulfato de Amônio , Densitometria , Óxido de Deutério , Fosfatos , Compostos de Potássio , Espalhamento de Radiação , Cloreto de Sódio , TemperaturaRESUMO
In a previous paper (Vivarès, D.; Bonneté, F. Acta Crystallogr., Sect. D 2002, 58, 472), protein-protein interactions of Aspergillus flavus urate oxidase (Uox) in solution were determined by small-angle X-ray scattering in the presence of different poly(ethylene glycol)s (PEG) in order to correlate second virial coefficient measurements with crystallization conditions. In this paper, we have characterized the experimental phase diagram of urate oxidase in the case of PEG 8000 by determining the solubility curve and the dilute part of the liquid-liquid phase separation (LLPS). Within this phase diagram, different mechanisms of urate oxidase crystal growth and LLPS can be observed by optical video microscopy. The influence of the LLPS on both the mechanisms and kinetics of urate oxidase crystal growth was observed by optical microscopy and small-angle X-ray scattering (SAXS). Interactions between the macromolecules were studied by SAXS in the dilute and dense phases of the demixed solution. It was observed that the LLPS precedes and slows down the crystallization. This study shows that urate oxidase is a good model to study protein/PEG mixtures in the general context of protein crystallization.
RESUMO
The mass densities, total cold neutron cross sections and small angle scattering of concentrated NaCl and KCl solutions in H2O or D2O (2H2O) were measured at 20 degrees C. The partial specific volumes of both salts increase with salt concentration and are significantly smaller in D2O than in H2O, showing that these salt solutions cannot be considered as isomorphous in H2O and D2O. As salt concentration increases for both salts, the total coherent cross sections for neutrons of the solutions also increase while the coherent small angle scattering decreases-observations that are consistent, respectively, with increasing correlations involving the ion and water components and a decrease in the particle number density and/or concentration fluctuations, in the solutions. Changes in incoherent scattering with salt concentration are essentially those expected from the solution compositions and densities.
RESUMO
Among lens crystallins, gamma-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of gamma-crystallins. At pH 7.0, all the gamma-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human gammaS (hgammaS) and bovine gammaS (bgammaS) formed disulfide-linked dimers, whereas the other bgamma-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hgammaS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric gammaS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The gammaS-crystallins lack the temperature-induced opacification observed with the other gamma-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.
Assuntos
Cristalinas/química , Cristalino/química , Sequência de Aminoácidos , Animais , Bovinos , Temperatura Baixa , Dimerização , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05-1.20 A) and neutron diffraction data (1.9-2.5 A) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H(2)O and D(2)O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.
Assuntos
Aspergillus flavus/enzimologia , Cristalografia/métodos , Difração de Nêutrons/métodos , Urato Oxidase/ultraestrutura , Difração de Raios X/métodos , Conformação Proteica , PrótonsRESUMO
The determination of the three-dimensional structures of biological macromolecules by X-ray diffraction generally requires large good-quality crystals, which are often difficult to obtain as crystal nucleation and growth depend upon a great number of physicochemical parameters. In the future, the emergence of structural genomic projects will require new and rapid methods to determine crystallization conditions. Until now, the prediction of crystallization conditions has essentially been based on the knowledge of interparticular interactions in solutions inferred from studies on small soluble proteins in the presence of salts. The present study, by small-angle X-ray scattering, of urate oxidase from Aspergillus flavus, a homotetrameric enzyme of 128kDa, allowed the extension of the results to the crystallization of large proteins in the presence of polyethylene glycol (PEG). The protein crystallization, the nucleation rate and the different morphological crystal shapes obtained were correlated with the second virial coefficient (A(2)), which was found to be in a restricted range at the low end of the 'crystallization slot' proposed by George & Wilson [(1994). Acta Cryst. D50, 361--365].
Assuntos
Aspergillus flavus/enzimologia , Polietilenoglicóis/química , Urato Oxidase/química , Aspergillus flavus/química , Simulação por Computador , Cristalização , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Conformação Proteica , Sais , TemperaturaRESUMO
It has been shown for several years that the second virial coefficient, A(2), can be helpfully used to describe the thermodynamic behavior of biological macromolecules in solution prior to crystallization. The coefficient, which reflects either repulsive or attractive interactions between particles, can allow a rapid determination of crystallization conditions. Different biological systems, from 14 kDa to 4600 kDa, were studied by small angle X-ray scattering. With large macromolecules, the A(2) values were found at the low end of the crystallization slot described by George & Wilson [(1994) Acta Cryst. D50, 361-365]. This led us to investigate the physical meaning of the second virial coefficient and to propose the use of the dimensionless second virial coefficient independent of the molecular weight and the size of the particle, which only takes into account the interaction potential between macromolecules, to predict successful crystallization conditions for large macromolecules. With this normalized coefficient (a(2)), the effect of salt on small proteins becomes equivalent to the effect of PEG on large macromolecules in terms of interaction potentials.
Assuntos
Cristalização/métodos , Animais , Bromovirus/química , Cristalização/estatística & dados numéricos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Peso Molecular , Muramidase/química , Polietilenoglicóis , Espalhamento de Radiação , Soluções , Termodinâmica , Urato Oxidase/química , Raios X , gama-Cristalinas/químicaRESUMO
We present the experimental and theoretical background of a method to characterize the protein-protein attractive potential induced by one of the mostly used crystallizing agents in the protein-field, the poly(ethylene glycol) (PEG). This attractive interaction is commonly called, in colloid physics, the depletion interaction. Small-Angle X-ray Scattering experiments and numerical treatments based on liquid-state theories were performed on urate oxidase-PEG mixtures with two different PEGs (3350 Da and 8000 Da). A "two-component" approach was used in which the polymer-polymer, the protein-polymer and the protein-protein pair potentials were determined. The resulting effective protein-protein potential was characterized. This potential is the sum of the free-polymer protein-protein potential and of the PEG-induced depletion potential. The depletion potential was found to be hardly dependent upon the protein concentration but strongly function of the polymer size and concentration. Our results were also compared with two models, which give an analytic expression for the depletion potential.
RESUMO
Osmotic pressure, small-angle X-ray scattering and quasi-elastic light scattering were used to study the medium-range interaction potentials between macromolecules in solution. These potentials determine macromolecular crystallization. Calf eye lens gamma-crystallins were used as a model system with the charge, and therefore the interactions, varied with pH. The second virial coefficient was determined under the same conditions with each of the three techniques. Osmotic pressure and quasi-elastic light scattering can be used conveniently in the laboratory to rapidly test the type of interactions (either attractive or repulsive) present in the solution. The measurement is direct with osmotic pressure, whereas with quasi-elastic light scattering, the directly measured coefficient is a combination of thermodynamic and hydrodynamic terms. X-rays, which require more sophisticated equipment such as synchrotron radiation facilities, can provide more detailed information on the interparticle potentials when models are used. At low ionic strength, two potentials were found necessary to account for the temperature and pH phase diagram as a function of protein concentration. The first potential is the van der Waals attractive potential that was previously shown to account for the fluid-fluid phase separation at low temperature. The second potential is an electrostatic coulombic repulsive potential which is a function of the protein charge and thus of the pH. The interaction trail could be followed at protein concentrations as low as 10 mg ml(-1). The results as a whole are expected to be valid for all compact low molecular weight proteins at low ionic strength.
RESUMO
In the field of protein crystallization, a better knowledge of the nucleation process is essential to control the nucleation rate, the growth and therefore the size and the quality of crystals. With that aim, it becomes clear that the important stage is the determination of the protein phase diagram. We highlighted and investigated the bovine pancreatic trypsin inhibitor (BPTI) binary liquid-liquid phase separation in 350 mM KSCN solutions as a function of temperature. We measured the low concentration part of the binodal curve using light scattering and optical microscopy. We show, from small angle X-ray scattering experiments, that the high concentrated phase sediments in the bottom of the capillary and we analysed the low concentrated phase in terms of monomers/decamers equilibrium.
Assuntos
Aprotinina/química , Aprotinina/isolamento & purificação , Animais , Bovinos , Cristalização , Luz , Espalhamento de Radiação , Soluções , Tiocianatos , Raios XRESUMO
A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles.