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

The G98R mutant of αA-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, D⁷¹FVIFLDVKHFSPEDLTVK⁸⁸ (αA-minichaperone or mini-αA) prevented aggregation of αAG98R. However, the mechanism of stabilization of αAG98R from aggregation is not fully understood. The purpose of this study was to determine whether the surface charge (zeta (ζ) potential) of αAG98R in the presence of the peptide chaperone contributed to the stabilization of mutant protein, and to identify the sites of interaction between αAG98R and the peptide chaperone.

Wild-type αA-crystallin (αAWT) and recombinant mutant αAG98R were purified from Escherichia coli BL21(DE3)pLysS cells. The ζ potential values of αA-crystallins in the presence or absence of αA-minichaperone and purified protein-peptide complexes were estimated in a ζ potential analyzer. Potential regions within αAG98R that bind the αA-minichaperone were investigated by incubating the protein with a photoactivable minichaperone variant, followed by mass spectrometric analysis.

Binding of the αA-minichaperone to aggregation-prone αAG98R was accompanied by an increase in the ζ potential from -15.19±0.870 mV corresponding to αAG98R alone to -28.64±1.640 mV for the purified complex. Mass spectrometric analysis identified ¹MDVTIQHPWFK¹¹, ¹³TLGPFYPSR²¹, ⁵⁵TVLDSGISEVR⁶⁵, and ¹¹³EFHRR¹¹⁷ as the αA-minichaperone-binding regions in αAG98R. The results suggest the involvement of the N-terminal region and the α-crystallin domain in the peptide-mediated stabilization of αAG98R.

The αA-crystallin-derived minichaperone stabilizes αAG98R by compensating its lost surface charge. Methods for increasing the ζ potential of aggregating proteins can be a potential approach for therapy to protein aggregation diseases.