F9S mutation in Mice γS-crystallin
triggers protein unfolding: implication of cataract formation
Justin Wu
Department of Biochemistry
Transparency and refraction of lens depend directly on the integrity of the crystallin family proteins. Modification of these proteins both at congenital and post-translational level can cause formation of inclusion in lens and ultimately lead to cataract. Two key questions related to cataract formation still remain to be answered: 1) what leads to protein aggregate after modification, through simple precipitation or through increased conformational dynamics, and 2) what happens when crystallins unfold, forming light-scattering aggregates or cyto-toxic amyloid fibrils? To address these questions, the solution structure and their dynamics of both wild type and F9S mutated γS-crystallin have been investigated. While the structure of the mutant resembles its wild type counterpart, it demonstrates up to 100-fold increases in deuterium/hydrogen exchange rate within the entire amino-terminal domain, whereas no changes were observed in the C-terminal domain. Such contrast between the two domains becomes much more significant when temperature increases, implying that protein aggregation at physiological temperature of F9S may start from the unfolding of the N-terminal domain. Consistent with such hypothesis is that F9S displays two distinct transitions in tryptophan fluorescence spectrum in the presence of increasing amount of GuHCl.
Fluorescence measurement of the partially denatured mutant induced by 0.5M GuHCl or elevated temperature in the presence of Thioflavine T (ThT) indicated that the unfolded protein is amyloidogenic. Atomic force microscopy studies demonstrated that long fibers can be developed in the similar conditions. Such fibrils can significantly shorten the lag phase of formation of ThT-sensitive fibrils. This observation is rather intriguing because similar fibril structures were present in F9S cataractous lens. Therefore, amyloid fibrils from unfolded crystallines may trigger the cascade of cataract formation.