Mechanism of Suppression of Protein Aggregation by α-Crystallin

This review summarizes experimental data illuminating the mechanism of suppression of heat-induced protein aggregation by a-crystallin, one of the small heat shock proteins. The dynamic light scattering data show that the initial stage of thermal aggregation of proteins is the formation of the initial aggregates involving hundreds of molecules of the denatured protein. Further sticking of the starting aggregates proceeds in a regime of diffusion-limited cluster-cluster aggregation. The protective effect of a-crystallin is due to transition of the aggregation process to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower than unity

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 values of parameters of Eq. (20) for suppression of DTT-induced aggregation of BSA by chemical chaperones (<i>R</i>² is the coefficient of determination).

<p>The values of parameters of Eq. (20) for suppression of DTT-induced aggregation of BSA by chemical chaperones (<i>R</i>² is the coefficient of determination).</p

Iinitial rate of DDT-induced aggregation of BSA at 45°C as a function of cross-linked α-crystallin concentration.

<p>The solid line was calculated from Eq. (12) at <i>S</i>₀ = 4.7 subunits of α-crystallin per one BSA molecule. The dotted line corresponds to the dependence of (<i>K</i>_agg/<i>K</i>_agg,0)^1/<i>n</i> on concentration of intact α-crystallin (<i>n</i> = 1.6).</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

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

SEC elution profiles of cross-linked and intact α-crystallin on a TSK-gel HW-55f column.

<p>The fraction of cross-linked protein marked with gray color was isolated for further testing the chaperone-like activity. Triangles point out retention time of the protein standards: thyroglobulin (660 kDa), catalase (440 kDa), aldolase (158 kDa), BSA (67 kDa), α-crystallin (20 kDa).</p