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

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

Analysis of the kinetics 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 light scattering intensity on temperature obtained at the following concentrations of GAPDH: (1) 0.1, (2) 0.75 and (3) 1.5 mg/ml. Points are the experimental data. The solid curves were calculated from Eq. (4). (<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 GAPDH concentration, respectively.</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

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 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

Parameters of aggregation of GDH (0.12 mg/ml) in the presence of different ligands (0.1 M Na-phosphate buffer, pH 7.6).

<p>Parameters of aggregation of GDH (0.12 mg/ml) in the presence of different ligands (0.1 M Na-phosphate buffer, pH 7.6).</p

Effect of different agents on Ph<i>b</i> aggregation.

<p>The initial parts of the dependences of the light scattering intensity on temperature for aggregation of Ph<i>b</i> (0.5 mg/ml) in the presence of the following agents: (1) control, (2) 1 mM AMP, (3) 0.19 M HP-β-CD, (4) 1 M TMAO and (5) α-crystallin at the concentration of 1 mg/ml. Points are the experimental data. The solid curves were calculated from Eq. (4).</p

Estimation of the size of the protein aggregates formed in the course of GDH aggregation registered in the regime of heating at the rate of 1°C/min (0.1 M Na-phosphate buffer, pH 7.6).

<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 GDH concentrations: (1) 0.1, (2) 0.2, (3) 0.3 and (4) 0.4 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. (9) on the GDH concentration, respectively.</p