Effect of Neurodegenerative Mutations in the NEFL Gene on Thermal Denaturation of the Neurofilament Light Chain Protein.

Effects of E90K, N98S, and A149V mutations in the light chain of neurofilaments (NFL) on the structure and thermal denaturation of the NFL molecule were investigated. By using circular dichroism spectroscopy, it was shown that these mutations did not lead to the changes in α-helical structure of NFL, but they caused noticeable effects on the stability of the molecule. We also identified calorimetric domains in the NFL structure by using differential scanning calorimetry. It was shown that the E90K replacement leads to the disappearance of the low-temperature thermal transition (domain 1). The mutations cause changes in the enthalpy of NFL domains melting, as well as lead to the significant changes in the melting temperatures (Tm) of some calorimetric domains. Thus, despite the fact that all these mutations are associated with the development of Charcot-Marie-Tooth neuropathy, and two of them are even located very close to each other in the coil 1A, they affect differently structure and stability of the NFL molecule.

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.

Effects of Molecular Crowding and Betaine on HSPB5 Interactions, with Target Proteins Differing in the Quaternary Structure and Aggregation Mechanism.

The aggregation of intracellular proteins may be enhanced under stress. The expression of heat-shock proteins (HSPs) and the accumulation of osmolytes are among the cellular protective mechanisms in these conditions. In addition, one should remember that the cell environment is highly crowded. The antiaggregation activity of HSPB5 and the effect on it of either a crowding agent (polyethylene glycol (PEG)) or an osmolyte (betaine), or their mixture, were tested on the aggregation of two target proteins that differ in the order of aggregation with respect to the protein: thermal aggregation of glutamate dehydrogenase and DTT-induced aggregation of lysozyme. The kinetic analysis of the dynamic light-scattering data indicates that crowding can decrease the chaperone-like activity of HSPB5. Nonetheless, the analytical ultracentrifugation shows the protective effect of HSPB5, which retains protein aggregates in a soluble state. Overall, various additives may either improve or impair the antiaggregation activity of HSPB5 against different protein targets. The mixed crowding arising from the presence of PEG and 1 M betaine demonstrates an extraordinary effect on the oligomeric state of protein aggregates. The shift in the equilibrium of HSPB5 dynamic ensembles allows for the regulation of its antiaggregation activity. Crowding can modulate HSPB5 activity by affecting protein-protein interactions.

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.

The mechanism of thermal aggregation of glutamate dehydrogenase. The effect of chemical chaperones.

Chemical chaperones are low-molecular compounds counteracting protein aggregation. Understanding of the mechanism of their effects is key to their potential use in biotechnology. The aggregation of bovine liver glutamate dehydrogenase (GDH) was studied at 40 °C and 50 °C using dynamic light scattering, analytical ultracentrifugation, size-exclusion chromatography and differential scanning calorimetry. At 40 °C the GDH aggregation proceeds through the slow stages of hexamer dissociation and formation of small oligomeric aggregates. At 50 °C these stages are transient. The rate-limiting stage of the overall aggregation process is unfolding of the protein molecule; the order of aggregation with respect to protein, n = 1. The test system based on GDH aggregation at 50 °C was used to quantify the anti-aggregation activity of chemical chaperones by comparing their half-saturation concentrations [L]0.5. Arginine ethyl ester had the highest anti-aggregation activity, with [L]0.5 = 4 ± 1 mM. For other additives, [L]0.5 was 22 ± 1 mM (arginine), 18 ± 1 mM (argininamide) and 95 ± 12 mM (proline). Arginine at concentrations up to 300 mM, argininamide at concentrations higher than 300 mM and arginine ethyl ester at concentrations higher than 500 mM enhance aggregate-aggregate sticking. These results explain the mechanism of heat-induced GDH aggregation and its peculiarities at different temperatures or in the presence of chemical chaperones.

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.

Structural and functional studies of D109A human αB-crystallin contributing to the development of cataract and cardiomyopathy diseases.

αB-crystallin (heat shock protein ß5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.

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.

3D structure of the native α-crystallin from bovine eye lens.

α-Crystallin is the major eye lens protein that has been shown to support lens transparency by preventing the aggregation of lens proteins. The 3D structure of α-crystallin is largely unknown. Electron microscopy, single-particle 3D reconstruction, size exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation were used to study the structure of the native α-crystallin. Native α-crystallin has a wide distribution in size. The shape of mass distribution is temperature-dependent, but the oligomers with a sedimentation coefficient of ~22 S (750-830 kDa) strongly prevailed at all temperatures used. A 3D model of native α-crystallin with resolution of ~2 nm was created. The model is asymmetrical, has an elongated bean-like shape 13 × 19 nm with a dense core and filamentous "kernel". It does not contain a central cavity. The majority of α-crystallin particles regardless of experimental conditions are 13 × 19 nm, which corresponds to 22S sedimentation coefficient, hydrodynamic diameter 20 nm and mass of 750-830 kD. These particles are in dynamic equilibrium with particles of smaller and larger sizes.

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.

Anti-aggregation activity of small heat shock proteins under crowded conditions.

It is becoming evident that small heat shock proteins (sHsps) are important players of protein homeostasis system. Their ability to bind misfolded proteins may play a crucial role in preventing protein aggregation in cells. The remarkable structural plasticity of sHsps is considered to underlie the mechanism of their activity. However, all our knowledge of the anti-aggregation functioning of sHsps is based on data obtained in vitro in media greatly different from the cellular highly crowded milieu. The present review highlights available data on the effect of crowding on the anti-aggregation activity of sHsps. There is some evidence that crowding affects conformation and dynamics of sHsps oligomers as well as their anti-aggregation properties. Crowding stimulates association of sHsp-client protein complexes into large-sized aggregates thus diminishing the apparent anti-aggregation activity of sHsps. Nevertheless, it is also shown that complexes between suboligomers (dissociated forms) of sHsps and client proteins may be stabilized and exist for longer period of time under crowded conditions. Moreover, crowding may retard the initial stages of aggregation which correspond to the formation of sHsp-containing nuclei and their clusters. Thus, dissociation of sHsps into suboligomers appears to be an important feature for the anti-aggregation activity of sHsps in crowded media.