Structural perturbation and enhancement of the chaperone-like activity of alpha-crystallin by arginine hydrochloride

Abstract

Structural perturbation of alpha-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens alpha-crystallin as well as recombinant human alphaA- and alphaB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for alphaB-crystallin than for alphaA-crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in alphaB-crystallin that is associated with desmin-related myopathy, results in a significant loss of chaperone-like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of alpha-crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far-UV CD spectra show no significant changes in secondary structure, whereas near-UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of alpha-crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of alpha-crystallin, as monitored by pyrene-solubilization and ANS-fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of alpha-crystallin and enhances its chaperone-like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications.

Figure 1.

Effect of various additives on the chaperone-like activity of α-crystallin (0.1mg/mL) toward DTT-induced aggregation of insulin (0.2mg/mL) at 37°C. (A) Insulin in the absence (open circles) and in the presence of calf eye lens α-crystallin (filled circles). (B) Effect of Arg.HCl. Aggregation of insulin in buffer containing 200 mM Arg.HCl (open triangles); aggregation of insulin in the presence of α-crystallin (0.1 mg/mL) and 200 mM Arg.HCl (filled triangles). Symbols represent different curve types and not data points. (Inset) Effect of Lys.2HCl. Insulin + 100 mM Lys2HCl (open circles), α-crystallin + Insulin + 100 mM Lys2HCl (filled circles), Insulin + 300 mM Lys2HCl (solid line), α-crystallin + insulin + 300 mM Lys2HCl (broken line). (C) Effect of various concentrations of Arg.HCl (open circles), Gdn.HCl (filled circles), and AGdn.HCl (triangles). (D) Effect of Arg.HCl on the chaperone-like activity of recombinant human αA- and αB-crystallins toward the DTT-induced aggregation of insulin at 37°C. Percent protection offered by 0.1 mg/mL αA-crystallin (open circles), and 0.05 mg/mL αB-crystallin (filled circles), toward the aggregation of insulin (0.2 mg/mL) as a function of Arg. HCl concentration.

Figure 2.

(A) Solubilization of pyrene by α-crystallin as a function of concentration of Arg.HCl at 37°C. S_α = A_α − A_b, S_αArg = A_αarg − A_bArg, where A_b, A_bArg, A_α, and A_αarg are the absorbance values at 338 nm of the pyrene solubilized in buffer alone, buffer containing arginine, α-crystallin, and α-crystallin in the presence of arginine, respectively. S_α represents the solubility of pyrene by α-crystallin and S_αArg represents the solubility of pyrene by α-crystallin in the presence of Arg.HCl. (B) Relative fluorescence intensity of ANS bound to α-crystallin in the presence of increasing concentrations of Arg.HCl at 37°C.

Figure 3.

Circular dichroism spectra of α-crystallin in the presence and in the absence of Arg HCl at 37°C. (A) Near-UV CD spectra. (B) Far-UV CD spectra. α-Crystallin in Buffer A alone (curve 1) and the buffer containing 100 mM (curve 2), 200 mM (curve 3), and 300 mM (curve 4) Arg.HCl. To record far UV-CD spectra, DL-Arg.HCl was used. ([ϑ]_MRM) Mean residue mass ellipticity.

Figure 4.

Sedimentation of the Arg.HCl-treated (filled circles) and untreated (open circles)α-crystallin through a linear glycerol gradient (10%–40%). The samples were incubated at 37°C for 2 h before loading on to the gradient. The positions of proteins used for standard molecular masses are also indicated. (a) Aldolase (158 kD); (b) catalase (232 kD); (c) thyroglobulin (669 kD). See Materials and Methods for details.