Contributions of aromatic pairs to the folding and stability of long‐lived human γD‐crystallin

Abstract

Human cD-crystallin (HcD-Crys) is a highly stable protein that remains folded in the eye lens for the majority of an individual's lifetime. HcD-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs with eight b-strands. Six aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in the b-hairpin sequences of the Greek keys. Ultraviolet damage to the aromatic residues in lens crystallins may contribute to the genesis of cataract. Mutant proteins with these aromatic residues substituted with alanines were constructed and expressed in E. coli. All mutant proteins except F115A and F117A had lower thermal stability than the WT protein. In equilibrium experiments in guanidine hydrochloride (GuHCl), all mutant proteins had lower thermodynamic stability than the WT protein. N-terminal domain (N-td) substitutions shifted the N-td transition to lower GuHCl concentration, but the C-terminal domain (C-td) transition remained unaffected. C-td substitutions led to a more cooperative unfolding/refolding process, with both the N-td and C-td transitions shifted to lower GuHCl concentration. The aromatic pairs conserved for each Greek key motif (Greek key pairs) had larger contributions to both thermal stability and thermodynamic stability than the other pairs. Aromatic-aromatic interaction was estimated as 1.5-2.0 kcal/mol. In kinetic experiments, N-td substitutions accelerated the early phase of unfolding, while C-td substitutions accelerated the late phase, suggesting independent domain unfolding. Only substitutions of the second Greek key pair of each crystallin domain slowed refolding. The second Greek keys may provide nucleation sites during the folding of the double-Greek-key crystallin domains.

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy

“…In this case, the aggregating protein was human ␥D-crystallin carrying a double-alanine substitution of tyrosine residues, Y92A/Y97A (18,25). Suppression of aggregation by WT CCT5 was similar to that found with WT human ␥D-crystallin (Fig.…”

Gamma crystallins of the human eye lens

“…Besides the tyrosine corners, six aromatic pairs (four tyr/tyr, one tyr/phe and one phe/phe) are present in the β-hairpin sequences of the Greek key. The role of these aromatic pairing to the stability of the molecule has been highlighted by Kong and King [41]. The role that trp residues (W) play is also worth commenting upon.…”

The βγ-crystallins: Native state stability and pathways to aggregation

“…These studies revealed the significant stabilizing effect of domain interface residues, including hydrophobic residues and glutamines (Flaugh et al, 2005b; Flaugh et al, 2006; Mills-Henry et al, 2007). Substitutions of tyrosine residues have elucidated the contributions of both conserved and variable tyrosine pairs (Kong and King, 2011). Tryptophan residues have been investigated both for their contribution to native-state stability (Kosinski-Collins et al, 2004; Xia et al, 2013); E.S., T. Takata, and J.…”