Abstract: Objective To find the molecular mechanism underlying the effect of congenital cataract related 129^th single-site mutation G129C on the thermal stability of human γC crystallin (HγC) protein structure. Methods HγC-WT and HγC-G129C were expressed and purified in vitro. The changes of intrinsic fluorescence intensity and static light scattering intensity of proteins with temperature were measured, and the temperature dependence of the folding and aggregation structures of HγC-WT and HγC-G129C was compared. Results When temperature was below 65 ℃, the barycentric mean of the intrinsic fluorescence of HγC-WT and HγC-G129C shifted towards a longer wavelength and the fluorescence intensity decreased with the increasing temperature, which was believed to be the evidence of unfolded protein conformation. When the temperature was higher than 65 ℃, the static light scattering intensity increased significantly with the temperature, indicating the protein aggregation upon heating. The wild-type HγC-G129C showed a stronger aggregation potency. During the thermal de-naturation process of HγC-WT and HγC-G129C, the crossing-point temperatures were 74.5 ℃ and 55.5 ℃, respectively. HγC-WT showed higher thermal stability. Conclusions The congenital cataract-associated G129C mutation significantly weakens conformational stability of γC-crystallin.

Key words: crystallin, thermal stability, aggregation, intrinsic fluorescence, static light scattering