Effects of osmolytes under crowding conditions on the properties of muscle glycogen phosphorylase b.

The study of the relationship between the activity and stability of enzymes under crowding conditions in the presence of osmolytes is important for understanding the functioning of a living cell. The effect of osmolytes (trehalose and betaine) on the secondary and tertiary structure and activity of muscle glycogen phosphorylase b (Phb) under crowding conditions created by PEG 2000 and PEG 20000 was investigated using dynamic light scattering, differential scanning calorimetry, circular dichroism spectroscopy, fluorimetry and enzymatic activity assay. At 25 °C PEGs increased Phb activity, but PEG 20000 to a greater extent. Wherein, PEG 20000 significantly destabilized its tertiary and secondary structure, in contrast to PEG 2000. Trehalose removed the effects of PEGs on Phb, while betaine significantly reduced the activating effect of PEG 20000 without affecting the action of PEG 2000. Under heat stress at 48 °C, the protective effect of osmolytes under crowding conditions was more pronounced than at room temperature, and the Phb activity in the presence of osmolytes was higher in these conditions than in diluted solutions. These results provide important insights into the complex mechanism, by which osmolytes affect the structure and activity of Phb under crowding conditions.

Effect of Chemical Chaperones on the Stability of Proteins during Heat- or Freeze-Thaw Stress.

The importance of studying the structural stability of proteins is determined by the structure-function relationship. Protein stability is influenced by many factors among which are freeze-thaw and thermal stresses. The effect of trehalose, betaine, sorbitol and 2-hydroxypropyl-ß-cyclodextrin (HPCD) on the stability and aggregation of bovine liver glutamate dehydrogenase (GDH) upon heating at 50 °C or freeze-thawing was studied by dynamic light scattering, differential scanning calorimetry, analytical ultracentrifugation and circular dichroism spectroscopy. A freeze-thaw cycle resulted in the complete loss of the secondary and tertiary structure, and aggregation of GDH. All the cosolutes suppressed freeze-thaw- and heat-induced aggregation of GDH and increased the protein thermal stability. The effective concentrations of the cosolutes during freeze-thawing were lower than during heating. Sorbitol exhibited the highest anti-aggregation activity under freeze-thaw stress, whereas the most effective agents stabilizing the tertiary structure of GDH were HPCD and betaine. HPCD and trehalose were the most effective agents suppressing GDH thermal aggregation. All the chemical chaperones stabilized various soluble oligomeric forms of GDH against both types of stress. The data on GDH were compared with the effects of the same cosolutes on glycogen phosphorylase b during thermal and freeze-thaw-induced aggregation. This research can find further application in biotechnology and pharmaceutics.

The Effect of Chemical Chaperones on Proteins with Different Aggregation Kinetics.

Formation and accumulation of protein aggregates adversely affect intracellular processes in living cells and are negative factors in the production and storage of protein preparations. Chemical chaperones can prevent protein aggregation, but this effect is not universal and depends on the target protein structure and kinetics of its aggregation. We studied the effect of betaine (Bet) and lysine (Lys) on thermal aggregation of muscle glycogen phosphorylase b (Phb) at 48°C (aggregation order, n = 0.5), UV-irradiated Phb (UV-Phb) at 37°C (n = 1), and apo-form of Phb (apo-Phb) at 37°C (n = 2). Using dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation, we have shown that Bet protected Phb and apo-Phb from aggregation, but accelerated the aggregation of UV-Phb. At the same time, Lys prevented UV-Phb and apo-Phb aggregation, but increased the rate of Phb aggregation. The mechanisms of chemical chaperone action on the tertiary and quaternary structures and kinetics of thermal aggregation of the target proteins are discussed. Comparison of the effects of chemical chaperones on the proteins with different aggregation kinetics provides more complete information on the mechanism of their action.

Effect of Trehalose on Oligomeric State and Anti-Aggregation Activity of αB-Crystallin.

αB-Crystallin (αB-Cr), one of the main crystalline lens proteins, along with other crystallins maintains lens transparency suppressing protein aggregation and thus preventing cataractogenesis. αB-Cr belongs to the class of molecular chaperones; being expressed in many tissues it has a dynamic quaternary structure, which is essential for its chaperone-like activity. Shift in the equilibrium between ensembles of oligomers of different size allows regulating the chaperone activity. Trehalose is known to inhibit protein aggregation in vivo and in vitro, and it is widely used in biotechnology. The results of studying the effect of trehalose on the chaperone-like activity of crystallins can serve as a basis for the design of drugs delaying cataractogenesis. We have studied the trehalose effect on the quaternary structure and anti-aggregation activity of αB-Cr using muscle glycogen phosphorylase b (Phb) as a target protein. According to the dynamic light scattering data, trehalose affects the nucleation stage of Phb thermal aggregation at 48°C, and an increase in the αB-Cr adsorption capacity (AC0) is the main effect of trehalose on the aggregation process in the presence of the protein chaperone (AC0 increases 1.5-fold in the presence of 66 mM trehalose). According to the sedimentation analysis data, trehalose stabilizes the dimeric form of Phb at the stages of denaturation and dissociation and enhances the interaction of αB-Cr with the target protein. Moreover, trehalose shifts the equilibrium between the αB-Cr oligomers towards the smaller forms. Thus, trehalose affects the quaternary structure of αB-Cr and increases its anti-aggregation activity at the nucleation stage.

Effect of Betaine and Arginine on Interaction of αB-Crystallin with Glycogen Phosphorylase b.

Protein-protein interactions (PPIs) play an important role in many biological processes in a living cell. Among them chaperone-client interactions are the most important. In this work PPIs of αB-crystallin and glycogen phosphorylase b (Phb) in the presence of betaine (Bet) and arginine (Arg) at 48 °C and ionic strength of 0.15 M were studied using methods of dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation. It was shown that Bet enhanced, while Arg reduced both the stability of αB-crystallin and its adsorption capacity (AC0) to the target protein at the stage of aggregate growth. Thus, the anti-aggregation activity of αB-crystallin increased in the presence of Bet and decreased under the influence of Arg, which resulted in inhibition or acceleration of Phb aggregation, respectively. Our data show that chemical chaperones can influence the tertiary and quaternary structure of both the target protein and the protein chaperone. The presence of the substrate protein also affects the quaternary structure of αB-crystallin, causing its disassembly. This is inextricably linked to the anti-aggregation activity of αB-crystallin, which in turn affects its PPI with the target protein. Thus, our studies contribute to understanding the mechanism of interaction between chaperones and proteins.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

Effect of arginine on stability and aggregation of muscle glycogen phosphorylase b.

Arginine (Arg) is frequently used in biotechnology and pharmaceutics to stabilize protein preparations. When using charged ions like Arg, it is necessary to take into account their contribution to the increase in ionic strength, in addition to the effect of Arg on particular processes occurring under the conditions of constancy of ionic strength. Here, we examined contribution of ionic strength (0.15 and 0.5 M) to the effects of Arg on denaturation, thermal inactivation and aggregation of skeletal muscle glycogen phosphorylase b (Phb). Dynamic light scattering, analytical ultracentrifugation, differential scanning calorimetry, circular dichroism and enzymatic activity assay were used to assess the effects of Arg at constant ionic strength compared with the effects of ionic strength alone. We found that high ionic strength did not affect the secondary structure of Phb, but changed conformation of the protein. Such a destabilization of the enzyme causes an increase in the initial rate of aggregation and inactivation of Phb thereby affecting its denaturation. Binding of Arg causes additional changes in the protein conformation, weakening the bonds between monomers in the dimer. This causes the dimer to dissociate into monomers, which rapidly aggregate. Thus, Arg acts on these processes much stronger than just ionic strength.

Chaperone-Like Activity of HSPB5: The Effects of Quaternary Structure Dynamics and Crowding.

Small heat-shock proteins (sHSPs) are ATP-independent molecular chaperones that interact with partially unfolded proteins, preventing their aberrant aggregation, thereby exhibiting a chaperone-like activity. Dynamics of the quaternary structure plays an important role in the chaperone-like activity of sHSPs. However, relationship between the dynamic structure of sHSPs and their chaperone-like activity remains insufficiently characterized. Many factors (temperature, ions, a target protein, crowding etc.) affect the structure and activity of sHSPs. The least studied is an effect of crowding on sHSPs activity. In this work the chaperone-like activity of HSPB5 was quantitatively characterized by dynamic light scattering using two test systems, namely test systems based on heat-induced aggregation of muscle glycogen phosphorylase b (Phb) at 48 °C and dithiothreitol-induced aggregation of α-lactalbumin at 37 °C. Analytical ultracentrifugation was used to control the oligomeric state of HSPB5 and target proteins. The possible anti-aggregation functioning of suboligomeric forms of HSPB5 is discussed. The effect of crowding on HSPB5 anti-aggregation activity was characterized using Phb as a target protein. The duration of the nucleation stage was shown to decrease with simultaneous increase in the relative rate of aggregation of Phb in the presence of HSPB5 under crowded conditions. Crowding may subtly modulate sHSPs activity.

Effect of Arginine on Chaperone-Like Activity of HspB6 and Monomeric 14-3-3ζ.

The effect of protein chaperones HspB6 and the monomeric form of the protein 14-3-3ζ (14-3-3ζm) on a test system based on thermal aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) at 37 °C and a constant ionic strength (0.15 M) was studied using dynamic light scattering. A significant increase in the anti-aggregation activity of HspB6 and 14-3-3ζm was demonstrated in the presence of 0.1 M arginine (Arg). To compare the effects of these chaperones on UV-Phb aggregation, the values of initial stoichiometry of the chaperone-target protein complex (S0) were used. The analysis of the S0 values shows that in the presence of Arg fewer chaperone subunits are needed to completely prevent aggregation of the UV-Phb subunit. The changes in the structures of HspB6 and 14-3-3ζm induced by binding of Arg were evaluated by the fluorescence spectroscopy and differential scanning calorimetry. It was suggested that Arg caused conformational changes in chaperone molecules, which led to a decrease in the thermal stability of protein chaperones and their destabilization.

Comparative effects of trehalose and 2-hydroxypropyl-ß-cyclodextrin on aggregation of UV-irradiated muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules which enhance folding and prevent aggregation of proteins. Investigation of their effects on the processes of protein aggregation is of importance for further understanding of implication of protein aggregation in neurodegenerative diseases, as well as for solving biotechnological tasks. The effects of chemical chaperones trehalose and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) at 37 °C have been studied. The process of thermal aggregation of UV-Phb includes a slow stage of structural reorganization of the UV-Phb molecule, nucleation stage and fast attachment of structurally reorganized UV-Phb molecules to nuclei formed during the nucleation stage. It was shown that both trehalose and HP-ß-CD increased the duration of the nucleation phase and slowed down the rate of structural reorganization of the UV-Phb molecule. This conclusion has been confirmed by the circular dichroism data. In the absence of chaperones, 82% UV-Phb aggregates, whereas in the presence of HP-ß-CD or trehalose the portion of aggregated protein decreases to 70 and 66%, respectively. The data on analytical ultracentrifugation demonstrated that in the presence of these additives the size of protein aggregates decreased. Analysis of the combined effect of trehalose and HP-ß-CD on UV-Phb aggregation showed that protein aggregation was independently affected by trehalose and HP-ß-CD.

Kinetic regime of Ca2+ and Mg2+-induced aggregation of phosphorylase kinase at 40 °C.

Many functions of phosphorylase kinase (PhK) are regulated by Ca2+ and Mg2+ ions. Ca2+ and Mg2+ ions stimulate activity of PhK, induce the changes in the tertiary and quaternary structure of the hexadecameric enzyme molecule, provoke association/aggregation of PhK molecules, enhance PhK binding to glycogen. To establish the kinetic regime of Ca2+ and Mg2+-induced aggregation of PhK from rabbit skeletal muscles at 40 °C, in the present work the kinetics of aggregation was studied at various protein concentrations using the dynamic light scattering. The proposed mechanism of aggregation involves the stage of unfolding of the protein molecule with retention of the integrity of its oligomeric structure, the nucleation stage and stages of the growth of protein aggregates. The initial rate of the aggregation process at the stage of aggregate growth depends linearly on the protein concentration. This means that the order of aggregation with respect to the protein is equal to unity and the aggregation rate is limited by the rate of protein unfolding. The rate constant of the first order characterizing the stage of protein unfolding was found to be equal to 0.071 min-1 (40 mM Hepes, pH 6.8, 100 mM NaCl, 0.1 mM Ca2+, 10 mM Mg2+).

Oligomeric state of αB-crystallin under crowded conditions.

Small heat shock proteins (sHsps) are molecular chaperones preventing protein aggregation. Dynamics of quaternary structure plays an important role in the chaperone-like activity of sHsps. However, an interrelation between the oligomeric state and chaperone-like activity of sHsps remains insufficiently characterized. Most of the accumulated data were obtained in dilute protein solutions, leaving the question of the oligomeric state of sHsps in crowded intracellular media largely unanswered. Here, we analyzed the effect of crowding on the oligomeric state of αB-crystallin (αB-Cr) using analytical ultracentrifugation. Marked increase in the sedimentation coefficient of αB-Cr was observed in the presence of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and trimethylamine N-oxide (TMAO) at 48 °C. An especially pronounced effect was detected for the PEG and TMAO mixture, where the sedimentation coefficient (s20,w) of αB-Cr increased from 10.7 S in dilute solution up to 40.7 S in the presence of crowding agents. In the PEG + TMAO mixture, addition of model protein substrate (muscle glycogen phosphorylase b) induced dissociation of large αB-Cr oligomers and formation of complexes with smaller sedimentation coefficients, supporting the idea that, under crowding conditions, protein substrates can promote dissociation of large αB-Cr oligomers.

Effect of ionic strength and arginine on aggregation of UV-irradiated muscle glycogen phosphorylase b.

In this work the effect of ionic strength and arginine on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) was studied using dynamic light scattering at 37 °C at various ionic strengths (0.02-0.7 M). Under these conditions the rate-limiting stage of the overall aggregation process is the structural reorganization of UV-Phb, which can be characterized by the first order rate constant kI. It was shown that an increase in NaCl concentration caused a decrease in the kI value, suggesting a slowdown of the UV-Phb structural reorganization. Circular dichroism data confirmed this conclusion. Arginine is widely used in biotechnology as an agent suppressing protein aggregation. However, arginine is a charged molecule, and, when studying the action of arginine on protein aggregation, the effects of ionic strength should be taken into account. To evaluate the effect of arginine, experiments were conducted at fixed values of ionic strength (0.15 M and 0.5 M). It was shown that at a low ionic strength arginine (0-0.13 M) accelerated the process of protein aggregation, whereas at higher ionic strength arginine (0-0.48 M) acted as an aggregation suppressor.

Mechanism of aggregation of UV-irradiated glycogen phosphorylase b at a low temperature in the presence of crowders and trimethylamine N-oxide.

To characterize the initial stages of protein aggregation, the kinetics of aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) was studied under conditions when the aggregation proceeded at a low rate (10°C, 0.03M Hepes buffer, pH6.8, containing 0.1M NaCl). Aggregation of UV-Phb was induced by polyethylene glycol and Ficoll-70, acting as crowders, or a natural osmolyte trimethylamine N-oxide (TMAO). It has been shown that the initial rate of the stage of aggregate growth is proportional to the protein concentration squared, suggesting that the order of aggregation with respect to the protein is equal to two. It has been concluded that the aggregation mechanism of UV-Phb at 10°C in the presence of crowders includes the nucleation stage and stages of protein aggregate growth (the basic aggregation pathway). The aggregation mechanism is complicated in the presence of TMAO, and the stage of aggregate-aggregate assembly induced by TMAO should be added to the basic aggregation pathway. It has been shown that the ability of TMAO at a low concentration (0.05M) to induce aggregation of UV-Phb is due to the decrease in the absolute value of zeta potential of the protein in the presence of TMAO.

A thermal after-effect of UV irradiation of muscle glycogen phosphorylase b.

Different test systems are used to characterize the anti-aggregation efficiency of molecular chaperone proteins and of low-molecular-weight chemical chaperones. Test systems based on aggregation of UV-irradiated protein are of special interest because they allow studying the protective action of different agents at physiological temperatures. The kinetics of UV-irradiated glycogen phosphorylase b (UV-Phb) from rabbit skeletal muscle was studied at 37°C using dynamic light scattering in a wide range of protein concentrations. It has been shown that the order of aggregation with respect to the protein is equal to unity. A conclusion has been made that the rate-limiting stage of the overall process of aggregation is heat-induced structural reorganization of a UV-Phb molecule, which contains concealed damage.

Kinetic regime of thermal aggregation of holo- and apoglycogen phosphorylases b.

To characterize the role of pyridoxal 5'-phosphate in stabilization of the conformation of muscle glycogen phosphorylase b (Phb), the mechanism of thermal aggregation for holo- and apoforms of Phb has been studied using dynamic light scattering. The order of aggregation with respect to the protein (n) for aggregation of holoPhb at 48°C is equal to 0.5 suggesting that the dissociative mechanism of denaturation is operative and denaturation is followed by rapid aggregation stage. In the case of aggregation of apoPhb at 37°C n=2 and the rate-limiting stage is aggregation of unfolded protein molecules.

Checking for reversibility of aggregation of UV-irradiated glycogen phosphorylase b under crowding conditions.

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.

Effect of Ca2+ and Mg2+ ions on oligomeric state and chaperone-like activity of αB-crystallin in crowded media.

The main function of small heat shock proteins acting as suppressors of aggregation of non-native proteins is greatly influenced by crowded environment in the cell and the presence of divalent metal ions. The goal of the present work was to study the effects of Ca(2+) and Mg(2+) ions on the quaternary structure and anti-aggregation activity of αB-crystallin under crowding conditions. We showed that Ca(2+) and Mg(2+) ions induced formation of suboligomeric forms of αB-crystallin. This effect was retained in the presence of crowder (polyethylene glycol), although to a lesser degree. The chaperone-like activity of αB-crystallin was analyzed using heat-induced aggregation of myosin subfragment 1 (S1) at 40°C. In the absence of crowding agents chaperone-like activity of αB-crystallin exhibited low sensitivity to the presence of Ca(2+) and Mg(2+) ions. The addition of the crowding agents (polyethylene glycol 20000, Ficoll 70) dramatically increased S1 aggregation rates and significantly depressed anti-aggregation activity of αB-crystallin. Low concentrations of Ca(2+) (0.1mM) and Mg(2+) (10mM) partially restored the chaperone-like activity of αB-crystallin in the presence of crowders. This effect was observed at relatively low values of [αB-crystallin]/[S1] molar ratio, however, at [αB-crystallin]/[S1]>0.2 the stimulating effect of Ca(2+) became less pronounced. These findings might indicate that under crowded cell conditions different factors, including divalent cations, can effectively modulate chaperone-like activity of protein chaperones, which otherwise cannot properly cope with crowding-provoked accelerated rates of substrates aggregation.

Dual effect of arginine on aggregation of phosphorylase kinase.

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.