Abstract: Objective To reveal the thermally induced denaturation of wild-type human γD-crystallin(HGD) and congenital cataract-related mutant (HGD P23T), and compare the differences in the structural changes between wild-type and mutants during a heating process. Methods HGD and HGD P23T were expressed and purified. The temperature-dependent intrinsic fluorescence intensity and static light scattering intensity of the protein samples were measured to reveal the temperature-dependent folding and aggregation structural changes of HGD and HGD P23T. Results When the temperature was below 70 ℃, the barycentric mean of the intrinsic fluorescence of HGD and HGD P23T shifted towards a longer wavelength with increasing temperature and the fluorescence intensity decreased indicating the unfolded protein conformations. The conformational stability of HGD P23T was weaker than that of HGD. When temperature was higher than 70 ℃, the static light scattering intensity increased significantly with temperature, indicating protein aggregation upon heating. Relative to the wild-type,HGD P23T showed a stronger aggregation potency. Conclusions Heating disrupts the folding conformation of γD-crystallin, induces the unfolded protein to aggregate. The disease-associated P23T mutation significantly reduces the conformational stability of γD-crystallin.

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