Tatyana B. Eronina

Effect of proline on thermal inactivation, denaturation and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

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.

Evidence for the formation of start aggregates as an initial stage of protein aggregation.

The kinetics of thermal aggregation of glycogen phosphorylase b and glyceraldehyde 3‐phosphate dehydrogenase from rabbit skeletal muscles were studied using dynamic light scattering. Use of high concentrations of the enzymes (1–3 mg/ml) provided a simultaneous registration of the native enzyme forms and protein aggregates. It was shown that initially registered aggregates (start aggregates) were large‐sized particles. The hydrodynamic radius of the start aggregates was about 100 nm. The intermediate states between the native enzyme forms and start aggregates were not detected. The initial increase in the light scattering intensity is connected with accumulation of the start aggregates, the size of the latter remaining unchanged. From a certain moment in time aggregates of higher order, formed as a result of sticking of the start aggregates, make a major contribution to the enhancement of the light scattering intensity.

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.

Effect of α-crystallin on thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that the rmal aggregation of proteins proceeds in the kinetic regime where in the rate of aggregation is limited by diffusion of the interacting particles (the regime of “diffusion-limited cluster-cluster aggregation”). In the presence of α-crystallin, a prote in exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of “reaction-limited cluster-cluster aggregation”). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of α-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that α-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of the rmal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.

Influence of osmolytes on inactivation and aggregation of muscle glycogen phosphorylase b by guanidine hydrochloride. Stimulation of protein aggregation under crowding conditions.

The effects of the osmolytes trimethylamine-N-oxide (TMAO), betaine, proline, and glycine on the kinetics of inactivation and aggregation of rabbit skeletal muscle glycogen phosphorylase b by guanidine hydrochloride (GuHCl) have been studied. It is shown that the osmolytes TMAO and betaine exhibit the highest protective efficacy against phosphorylase b inactivation. A test system for studying the effects of macromolecular crowding induced by osmolytes on aggregation of proteins is proposed. TMAO and glycine increase the rate of phosphorylase b aggregation induced by GuHCl.

Interaction of Hsp27 with native phosphorylase kinase under crowding conditions

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.

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).

A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K agg(T−T 0)2, where K agg is the parameter characterizing the initial rate of aggregation and T 0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T 0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets.

Effect of 2-hydroxypropyl-β-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-β-cyclodextrin (HP-β-CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP-β-CD is destabilization of the protein molecule under action of HP-β-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-β-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-β-CD was substantiated by the data on the enzyme inhibition by HP-β-CD.

Effect of GroEL on Thermal Aggregation of Glycogen Phosphorylase b from Rabbit Skeletal Muscle

The suppression of the thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle by the chaperonin GroEL is studied using dynamic light scattering. It is shown that the decrease in the rate of Phb aggregation under the action of GroEL is due to the transition of the aggregation process from the kinetic regime, wherein the rate of aggregation is limited by diffusion of the interacting particles, to a regime where the sticking probability for the colliding particles becomes lower than one (reaction-limited cluster-cluster aggregation). The analytical-ultracentrifugation data show that elevated temperatures induce dissociation of the dimeric Phb. The formation of a complex between the denatured monomeric form of Phb and the dissociated forms of GroEL is detected during heating at 46 degrees C.