αA-crystallin-derived minichaperone stabilizes αAG98R-crystallin by affecting its zeta potential

Purpose: The G98R mutant of alpha A-crystallin is associated with the development of presenile cataracts. In vitro, the recombinant mutant protein exhibits altered structural and functional characteristics, along with the propensity to aggregate by itself and precipitate. Previously, we have reported that the N-terminal aspartate substituted form of the antiaggregation peptide, (DFVIFLDVKHFSPEDLTVK88)-F-71 (alpha A-minichaperone or mini-alpha A) prevented aggregation of alpha AG98R. However, the mechanism of stabilization of alpha AG98R from aggregation is not fully understood. The purpose of this study was to determine whether the surface charge (zeta (zeta) potential) of alpha AG98R in the presence of the peptide chaperone contributed to the stabilization of mutant protein, and to identify the sites of interaction between alpha AG98R and the peptide chaperone. Methods: Wild-type alpha A-crystallin (alpha AWT) and recombinant mutant alpha AG98R were purified from Escherichia coli BL21(DE3) pLysS cells. The zeta potential values of alpha A-crystallins in the presence or absence of alpha A-minichaperone and purified protein-peptide complexes were estimated in a zeta potential analyzer. Potential regions within alpha AG98R that bind the alpha A-minichaperone were investigated by incubating the protein with a photoactivable minichaperone variant, followed by mass spectrometric analysis. Results: Binding of the alpha A-minichaperone to aggregation-prone alpha AG98R was accompanied by an increase in the zeta potential from -15.19 +/- 0.870 mV corresponding to aAG98R alone to -28.64 +/- 1.640 mV for the purified complex. Mass spectrometric analysis identified (1)MDVTIQHPWFK(11), (13)TLGPFYPSR(21), (55)TVLDSGISEVR(65), and (EFHRR117)-E-113 as the alpha A-minichaperone-binding regions in alpha AG98R. The results suggest the involvement of the N-terminal region and the alpha-crystallin domain in the peptide-mediated stabilization of aAG98R. Conclusions: The alpha A-crystallin-derived minichaperone stabilizes alpha AG98R by compensating its lost surface charge. Methods for increasing the zeta potential of aggregating proteins can be a potential approach for therapy to protein aggregation diseases.