RESUMEN
It is believed that the initial stages of protein aggregation are reversible and can be reversed by simple dilution, whereas prolonged exposure to factors responsible for denaturing proteins (for example, to elevated temperatures) results in the formation of irreversible aggregates. A new approach has been developed to discriminate the stage of the formation of reversible aggregates. Aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) was studied at 10, 25 and 37 °C in the presence of crowders (polyethylene glycol and Ficoll-70) using dynamic light scattering and analytical ultracentrifugation (pH 6.8; 0.1M NaCl). The dilution of the protein solution in the course of aggregation at 10 °C results in the breakdown of protein aggregates suggesting that the aggregation process is reversible. When aggregation of UV-Phb is studied at 37 °C, reversibility is lacking. Chemical chaperones (arginine, proline) induce the breakdown of protein aggregates of UV-Phb formed at 10 °C. In the experiments carried out at 37 °C in the presence of crowder the addition of arginine results in disintegration of protein aggregates only at early stages of the aggregation process. It is assumed that general pathway of protein aggregation includes the formation of reversible, completely dissociable, partly dissociable and irreversible aggregates.
Asunto(s)
Glucógeno Fosforilasa de Forma Muscular/química , Agregado de Proteínas/efectos de los fármacos , Agregado de Proteínas/efectos de la radiación , Rayos Ultravioleta , Animales , Ficoll/farmacología , Polietilenglicoles/farmacología , Conejos , TemperaturaRESUMEN
Arginine is widely used in biotechnology as a folding enhancer and aggregation suppressor. However, its action on the stability of complexly organized oligomeric proteins, on the one hand, and its role in the formation of supramolecular structures, on the other hand, are poorly known. The investigation is concerned with the effects of arginine on protein-protein interactions using phosphorylase kinase (PhK) as an example. PhK, a 1.3MDa (αßγδ)4 hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme that catalyzes phosphorylation and activation of glycogen phosphorylase b. On the basis of light scattering measurements it was shown that arginine induced aggregation of Ca(2+)-free PhK. On the contrary, when studying Ca(2+), Mg(2+)-induced aggregation of PhK at 37°C, the protective effect of arginine was demonstrated. The data on analytical ultracentrifugation are indicative of disruption of PhK hexadecameric structure under the action of arginine. Though HspB6 and HspB5 suppress aggregation of PhK they do not block the disruption effect of arginine with respect to both forms of PhK (Ca(2+)-free and Ca(2+), Mg(2+)-bound conformers). The dual effect of arginine has been interpreted from view-point of dual behaviour of arginine, functioning both like an osmolyte and a protein denaturant.
Asunto(s)
Arginina/farmacología , Fosforilasa Quinasa/química , Agregado de Proteínas/efectos de los fármacos , Animales , Calcio/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Humanos , Hidrodinámica , Iones , Cinética , Magnesio/farmacología , Metilaminas/química , Fosforilasa Quinasa/metabolismo , Sustancias Protectoras/farmacología , Conejos , Temperatura , UltracentrifugaciónRESUMEN
The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).
Asunto(s)
Apoproteínas/metabolismo , Calor , Sustancias Macromoleculares/farmacología , Chaperonas Moleculares/farmacología , Fosforilasa b/metabolismo , Agregado de Proteínas/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Animales , Área Bajo la Curva , Bovinos , Reactivos de Enlaces Cruzados/farmacología , Cinética , Polietilenglicoles/farmacología , Prolina/farmacología , Conejos , alfa-Cristalinas/farmacologíaRESUMEN
The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin-target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5â=â53 and 58 mM, respectively).
Asunto(s)
Ditiotreitol/farmacología , Chaperonas Moleculares/metabolismo , Albúmina Sérica Bovina/química , Animales , Arginina/farmacología , Bovinos , Cromatografía en Gel , Reactivos de Enlaces Cruzados/farmacología , Electroforesis en Gel de Poliacrilamida , Fraccionamiento de Campo-Flujo , Cinética , Luz , Chaperonas Moleculares/química , Tamaño de la Partícula , Prolina/farmacología , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Desplegamiento Proteico/efectos de los fármacos , Refractometría , Dispersión de Radiación , Albúmina Sérica Bovina/metabolismo , Ultracentrifugación , Viscosidad/efectos de los fármacos , alfa-Cristalinas/química , alfa-Cristalinas/metabolismoRESUMEN
An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-ß-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-ß-CD reveals a purely protective effect.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Músculo Esquelético/enzimología , Rayos Ultravioleta , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Cinética , Chaperonas Moleculares/química , Desnaturalización Proteica , Estabilidad Proteica , Conejos , Temperatura , alfa-Cristalinas/química , beta-Ciclodextrinas/químicaRESUMEN
The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.
Asunto(s)
Fosforilasa b/metabolismo , alfa-Cristalinas/metabolismo , Animales , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Masculino , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fosforilasa b/efectos de la radiación , Desnaturalización Proteica , Conejos , Dispersión de Radiación , Ultracentrifugación , Rayos Ultravioleta , alfa-Cristalinas/químicaRESUMEN
The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP-ß-CD is destabilization of the protein molecule under action of HP-ß-CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP-ß-CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP-ß-CD was substantiated by the data on the enzyme inhibition by HP-ß-CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986-993, 2010.
Asunto(s)
Glucógeno Fosforilasa de Forma Muscular/química , Glucógeno Fosforilasa de Forma Muscular/efectos de los fármacos , beta-Ciclodextrinas/farmacología , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Estabilidad de Enzimas/efectos de los fármacos , Glucógeno Fosforilasa de Forma Muscular/metabolismo , Técnicas In Vitro , Cinética , Luz , Músculo Esquelético/enzimología , Multimerización de Proteína/efectos de los fármacos , Conejos , Dispersión de Radiación , TermodinámicaRESUMEN
Interaction of the wild type (wt) heat shock protein Hsp27 and its three-dimensional (3D) mutant (mimicking phosphorylation at Ser15, 78, and 82) with rabbit skeletal muscle phosphorylase kinase (PhK) has been studied under crowding conditions modeled by addition of 1 M trimethylamine N-oxide (TMAO). According to the data of sedimentation velocity and dynamic light scattering, crowding provokes the formation of large-sized associates of both PhK and Hsp27. Under crowding conditions, small associates of PhK and Hsp27 interact with each other thus leading to dissociation of large homooligomers of each protein. Taking into account high concentrations of PhK in the cell, we speculate that native PhK might modulate the oligomeric state and chaperone-like activity of Hsp27.
Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Sustancias Macromoleculares/metabolismo , Fosforilasa Quinasa/metabolismo , Animales , Fraccionamiento Químico , Humanos , Proteínas Mutantes/metabolismo , Unión Proteica , Conejos , UltracentrifugaciónRESUMEN
Effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of creatine kinase from rabbit skeletal muscle (RMCK) at 48 degrees C has been studied using dynamic light scattering. An increase in the duration of the lag period on the kinetic curves of aggregation, registered as an increment of the light scattering intensity in time, has been observed in the presence of HP-beta-CD. It has been shown that the initial parts of the dependences of the hydrodynamic radius (R(h)) of the protein aggregates on time follow the exponential law. The reciprocal value of parameter t(2R) (t(2R) is the time interval over which the R(h) value is doubled) was used to characterize the rate of aggregation. A 10-fold decrease in the 1/t(2R) value was observed in the presence of 76mM HP-beta-CD. Judging from the data on the kinetics of RMCK inactivation and the data on differential scanning calorimetry of RMCK, HP-beta-CD does not affect the rate of RMCK unfolding.
Asunto(s)
Creatina Quinasa/química , Creatina Quinasa/metabolismo , Músculo Esquelético/enzimología , Temperatura , beta-Ciclodextrinas/farmacología , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Cinética , Luz , Tamaño de la Partícula , Unión Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Conejos , Dispersión de Radiación , Espectrometría de FluorescenciaRESUMEN
The effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle at 45 degrees C has been studied using dynamic light scattering. In the presence of HP-beta-CD higher values of the rate of aggregation and larger aggregates were registered. The acceleration of GAPDH aggregation was due to destabilization of the enzyme molecule under the action of HP-beta-CD. This is evidenced by the data on thermal inactivation of GAPDH and differential scanning calorimetry.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Temperatura , beta-Ciclodextrinas/farmacología , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Activación Enzimática/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Conejos , Refractometría , Viscosidad/efectos de los fármacosRESUMEN
It has been shown that the relatively low concentrations of proline (0.1 M) have a slight accelerating effect on thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle registered by the accumulaton of the aggregated protein. The suppression of Phb aggregation at high proline concentrations is mainly due to the protective action of proline on the stage of unfolding of the Phb molecule. The enhancement of Phb stability in the presence of the high concentrations of proline was demonstrated by the data on differential scanning calorimetry, analytical ultracentrifugation and thermoinactivation kinetics. The construction of the protein aggregate size versus time plots allowed the acceleration of the stage of Phb aggregation in the presence of high concentrations of proline to be demonstrated. The obtained results are consistent with the predictions of the crowding theory.
Asunto(s)
Glucógeno Fosforilasa de Forma Muscular/química , Glucógeno Fosforilasa de Forma Muscular/metabolismo , Calor , Músculo Esquelético/enzimología , Prolina/farmacología , Animales , Rastreo Diferencial de Calorimetría , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Prolina/metabolismo , Unión Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Conejos , Dispersión de Radiación , UltracentrifugaciónRESUMEN
Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.
Asunto(s)
Músculo Esquelético/enzimología , Fosforilasa Quinasa/metabolismo , Animales , Concentración Osmolar , Fosforilasa Quinasa/aislamiento & purificación , Conejos , Dispersión de Radiación , UltracentrifugaciónRESUMEN
Self-association of phosphorylase kinase (PhK) and its interaction with glycogen (M=5500 kDa) and phosphorylase b (Phb) has been studied using analytical ultracentrifugation and turbidimetry under the conditions of molecular crowding arising from the presence of high concentrations of osmolytes. In accordance with the predictions of the molecular crowding theory, trimethylamine N-oxide (TMAO) and betaine greatly favor self-association of PhK induced by Mg2+ and Ca2+ and PhK interaction with glycogen. In contrast, proline suppresses these processes, probably, due to its specific interaction with PhK. All osmolytes tested prevented the complex formation between PhK and its physiological substrate, Phb. The specific interactions of PhK and Phb with glycogen, in the living cell, presumably is a factor allowing the negative effect of crowding on the recognition of Phb by PhK to be overcome.