Functional and Structural Properties of Cytoplasmic Tropomyosin Isoforms Tpm1.8 and Tpm1.9.

The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C-terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex.

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.

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.

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.

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.

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.

Chaperone-like activity of monomeric human 14-3-3ζ on different protein substrates.

Members of the 14-3-3 protein family interact with hundreds of different, predominantly phosphorylated, proteins. 14-3-3 dimers are prevalent but exist at the equilibrium with the monomers. Our previous studies using the engineered monomeric 14-3-3ζ (14-3-3ζm) showed that 14-3-3ζ monomer retained binding activity towards selected phosphorylated partners and, in addition, it prevented heat-induced aggregation of myosin subfragment 1. Since the chaperone-like activity of 14-3-3 monomers has been insufficiently studied, here we have analyzed the effect of 14-3-3ζm on the aggregation of different model proteins. We found that 14-3-3ζm demonstrated considerable chaperone-like activity by inhibiting the DTT-induced aggregation of insulin and thermally-induced aggregation of alcohol dehydrogenase and phosphorylase kinase. Importantly, the anti-aggregating activity of 14-3-3ζm was concentration-dependent and overall, was more pronounced than that of its dimeric counterpart. In some cases, the chaperone-like effect of 14-3-3ζm was comparable, or even higher, than that of the small heat shock proteins, HspB6 and HspB5. We suggest that 14-3-3s not only can bind and regulate the activity of multiple phosphoproteins, but also possess moonlighting chaperone-like activity, which is especially pronounced in the case of monomeric forms of 14-3-3 which can be present under certain stress conditions.

Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones.

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).

Polyamines putrescine and spermidine as modulators of protein aggregation rate: the effect on DTT-induced aggregation of α-lactalbumin.

Protein aggregation is undesirable for cells due to its possible toxicity, and is also undesirable in biotechnology and pharmaceuticals. Polyamines are known to be capable of both suppressing and stimulating protein aggregation. In the present work polyamines (spermidine, putrescine) have been shown to alter the pathway of α-lactalbumin aggregation induced by dithiothreitol, leading to the formation of larger protein particles during the initial stages of aggregation and promoting the later stage of sticking of aggregates. According to the aggregation kinetics data, polyamines accelerate protein aggregation in a concentration-dependent manner, with a maximum at 50 mM spermidine and 100 mM putrescine. With a further increase in polyamines concentration the effect of aggregation acceleration decreased, thus, the modulation of the aggregation rate by polyamines was shown. A comparison of the aggregation kinetics and hydrodynamic radii growth data registered by dynamic light scattering with the data obtained by asymmetric flow field-flow fractionation and analytical ultracentrifugation allowed us to describe the early stages of aggregation and formation of initial α-lactalbumin clusters. Our results provide a deeper insight into the mechanism of amorphous aggregation of α-lactalbumin and polyamines action on protein aggregation and protein-protein interaction in general.

Does the crowded cell-like environment reduce the chaperone-like activity of α-crystallin?

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.

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+).

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.

Dissociative mechanism for irreversible thermal denaturation of oligomeric proteins.

Protein stability is a fundamental characteristic essential for understanding conformational transformations of the proteins in the cell. When using protein preparations in biotechnology and biomedicine, the problem of protein stability is of great importance. The kinetics of denaturation of oligomeric proteins may have characteristic properties determined by the quaternary structure. The kinetic schemes of denaturation can include the multiple stages of conformational transitions in the protein oligomer and stages of reversible dissociation of the oligomer. In this case, the shape of the kinetic curve of denaturation or the shape of the melting curve registered by differential scanning calorimetry can vary with varying the protein concentration. The experimental data illustrating dissociative mechanism for irreversible thermal denaturation of oligomeric proteins have been summarized in the present review. The use of test systems based on thermal aggregation of oligomeric proteins for screening of agents possessing anti-aggregation activity is discussed.

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.

Effect of crowding and chaperones on self-association, aggregation and reconstitution of apophosphorylase b.

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5'-phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (α-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 °C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 °C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and α-crystallin, the complexes between unfolded apoPhb and dissociated forms of α-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of α-crystallin under crowding conditions.

Concentration dependence of chaperone-like activities of α-crystallin, αB-crystallin and proline.

Chaperone-like activities of α-crystallin, αB-crystallin and proline were studied using a test system based on aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation (v(agg)) of UV-irradiated Phb on the concentration of α-crystallin or αB-crystallin is indicative of the existence of two types of chaperone-protein substrate complexes differing significantly in affinity between the components of the complex. The dependence of v(agg) on the proline concentration is sigmoid (Hill coefficient is equal to 1.6) suggesting that the positive cooperative interactions between the proline molecules bound on the surface of the protein particles occur. When studying the combined suppressive action of α-crystallin and proline on aggregation of UV-irradiated Phb, a slight antagonism between proline used at a fixed concentration (0.15M) and α-crystallin was observed. At higher concentration of proline (0.5M) each chaperone acts independent of one another.

Effect of 2-hydroxypropyl-ß-cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

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.