Contributions of Aromatic Residues to the Folding and Stability of Human γd-Crystallin

Abstract

Human γD-crystallin (HγD-Crys), one of the most abundant proteins in the eye lens, exhibits two crystallin domains, each containing two Greek key motifs. HγD-Crys must remain folded and soluble throughout the human lifetime. Aggregation of crystallins leads to cataract.14 tyrosines and 6 phenylalanines reside in the 173-amino-acids HγD-Crys. 16 out of these 20 residues have aromatic partners within ∼4 A. “Tyrosine corner” is a structural element that bridges β-strands by hydrogen bonding the tyrosine hydroxyl group and a backbone carboxyl group. Also found are interacting tyrosine/phenylalanine pairs located at the β-hairpins. These aromatic structural elements may be important in the stability and/or the folding pathways.Site-specific mutants of the all the tyrosines and phenylalanines to alanines were constructed. Equilibrium and kinetic experiments were performed to assess stability and unfolding/refolding rates.For stability, all the N-terminal domain (N-td) mutants had the N-td destabilized, but C-td unaffected, with increased population of the single-domain-folded intermediate, although the extents of destabilization were different. All the C-td mutants had both the N-td and C-td destabilized, showing a more cooperative folding process that was best fit to a two-state model, and similarly, the degrees of destabilization varied. Selected tyrosines were mutated to phenylalanines with very little effect on the N-td or the C-td stabilities. For kinetics, C-td mutants had accelerated unfolding rates, while N-td mutants had no effect. Y45A, Y50A Y133A, Y138A had slower refolding rates, while other mutants had no effects.The results were consistent with a sequential unfolding pathway, in which the N-td of HγD-Crys unfolds first, followed by the C-td. The aromatic residues were almost all important in the mature stability of HγD-Crys, while a subset of these aromatic residues were important determinants of unfolding/refolding rate.