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

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)

Kinetics of DTT-induced aggregation of BSA.

<p>(A) The dependences of the light scattering intensity (<i>I</i>) on time obtained at the following concentrations of BSA: (1) 0.5, (2) 1 and (3) 1.5 mg/ml (0.1 M phosphate buffer, pH 7.0; 45°C). The concentration of DTT was 2 mM. Points are the experimental data. Solid curves were calculated from Eq. (3). (B) The dependences of the hydrodynamic radius (<i>R</i>_h) of particles on time registered for heated BSA solutions. BSA concentrations were the following: (1) 0.5, (2) 1.5 and (3) 3 mg/ml.</p

Effect of ArgAd on DTT-induced aggregation of BSA ([BSA] = 1.0 mg/ml; 2 mM DTT).

<p>(A) The dependences of the light scattering intensity on time obtained at the following concentrations of ArgAd: (1) 0, (2) 75 and (3) 150 mM. Points are the experimental data. The solid curves were calculated from Eq. (5). At [ArgAd] = 75 mM the fitting procedure gave the following values of parameters: <i>k</i>_agg = 40.1 (counts/s) min⁻², <i>t</i>₀ = 12.1 min and <i>K</i> = 1.18·10⁻³ min⁻². The dotted line was calculated from Eq. (3) at <i>k</i>_agg = 40.1 (counts/s) min⁻² and <i>t</i>₀ = 12.1 min. (B) The dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on time obtained in the absence of ArgAd (1) and in the presence of 150 mM ArgAd (2). (C) The dependence of the <i>K</i>_agg/<i>K</i>_agg,0 ratio on the concentration of ArgAd. Points are the experimental data corresponding to the following concentrations of BSA: (1) 0.5, (2) 1 and (3) 2 mg/ml. The solid curve was calculated from Eq. (20). Inset shows the dependence of the duration of the lag period (<i>t</i>₀) on the concentration of ArgAd.</p

Analysis of combined action of α-crystallin and Arg.

<p>The dependences of (<i>k</i>_agg)^1/<i>n</i> on the [ α -crystallin]/[BSA] ratio in the absence (squares) and in the presence of 100 mM Arg (circles). Conditions: [BSA] = 1 mg/ml, [DTT] = 2 mM, 0.1 M Na-phosphate buffer pH 7.0, 45°C.</p

Fractograms of BSA (1 mg/ml) heated at 45°C in the presence of 2 mM DTT.

<p>The heating times were the following: 20 (A), 45 (B) and 90 (D) min. AF4 conditions were the same as described in legend to Fig. 11.</p

Kinetic parameters for DDT-induced aggregation of BSA (45°C; 2 mM DTT).

<p>(A) The dependence of parameter <i>k</i>_agg on BSA concentration. The solid curve was calculated from Eq. (9) at <i>n</i> = 1.6. Inset shows the dependence of <i>K</i>_agg on BSA concentration in the logarithmic coordinates. (B) The dependence of duration of the lag period (<i>t</i>₀) on BSA concentration.</p