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 = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 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 T0 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

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 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-β-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

Thermal denaturation of Ph<i>b</i> (0.08 M Hepes-buffer, pH 6.8, containing 0.1 M NaCl).

<p>The dependences of the excess heat capacity () on temperature, obtained at the following concentrations of Ph<i>b</i>: (1) 0.95 and (2) 1.9 mg/ml. was calculated per dimer of Ph<i>b</i> with the molecular mass of 194.8 kDa. The heating rate was 1°C/min.</p

Effect of ADP on GDH aggregation.

<p>(<b>A</b>) The initial parts of the dependences of the light scattering intensity on temperature for aggregation of GDH (0.16 mg/ml) in the presence of the following concentrations of ADP: (1) 0, (2) 0.05, (3) 0.1, (4) 0.5 and (6) 2 mM. (<b>B</b> and <b>C</b>) The dependences of parameters <i>T</i>₀ and <i>K</i>_agg calculated from Eq. (4) on the ADP concentration, respectively. The solid curve in panel <b>C</b> was calculated from Eq. (10) at <i>K</i>_diss = 0.52 mM.</p

Estimation of the size of the protein aggregates formed in the course of GAPDH aggregation registered in the regime of heating at the rate of 1°C/min (10 mM Na-phosphate buffer, pH 7.5, containing 0.1 M NaCl).

<p>(<b>A</b>) The initial parts of the dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on temperature obtained at the following GAPDH concentrations: (1) 0.1, (2) 0.25, (3) 0.5 and (4) 1.5 mg/ml. (<b>B</b> and <b>C</b>) The dependences of parameter <i>R</i>_h,0 and the reciprocal value of parameter Δ<i>T</i>_2R calculated from Eq. (8) on the GAPDH concentration, respectively.</p

Effect of different agents on GDH aggregation.

<p>The initial parts of the dependences of the light scattering intensity on temperature for aggregation of GDH (0.12 mg/ml) in the presence of the following agents: (1) control; (2) 0.2 mM NADH; (3) 50 mM L-glutamate; (4) 0.2 mM NADH+0.5 mM ADP; (5) 50 mM L-leucine and (6) 0.5 mM ADP. Points are the experimental data. The solid curves were calculated from Eq. (4).</p

Quantification of anti-aggregation activity of chaperones: a test-system based on dithiothreitol-induced aggregation of bovine serum albumin.

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]0.5 = 53 and 58 mM, respectively)

The relationships between the increment of the light scattering intensity accompanying Ph<i>b</i> aggregation and the portion of the denatured Ph<i>b</i> (γ_den) calculated from the DSC data.

<p>Ph<i>b</i> concentrations were as follows: (1) 0.95 and (2) 1.9 mg/ml. The inset shows the initial parts of the curves. Points are the experimental data. The solid curves are calculated from Eq. (6).</p