Abstract
Human γD crystallin (HγD-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily β-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HγD-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in
Escherichia coli
exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type HγD-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37°C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type HγD-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of HγD-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The HγD-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of HγD-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo.