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Contributions of aromatic pairs to the folding and stability of long-lived human ?D-crystallin.

ABSTRACT: Human ?D-crystallin (H?D-Crys) is a highly stable protein that remains folded in the eye lens for the majority of an individual's lifetime. H?D-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs with eight ?-strands. Six aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in the ?-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.

SUBMITTER: Kong F

PROVIDER: S-EPMC3064831 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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

Kong Fanrong F King Jonathan J

Protein science : a publication of the Protein Society 20110301 3

Human γD-crystallin (HγD-Crys) is a highly stable protein that remains folded in the eye lens for the majority of an individual's lifetime. HγD-Crys exhibits two homologous crystallin domains, each containing two Greek key motifs with eight β-strands. Six aromatic pairs (four Tyr/Tyr, one Tyr/Phe and one Phe/Phe) are present in the β-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 ...[more]

PMID: 21432932

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Project description:Ultraviolet-radiation-induced damage to and aggregation of human lens crystallin proteins are thought to be a significant pathway to age-related cataract. The aromatic residues within the duplicated Greek key domains of ?- and ?-crystallins are the main ultraviolet absorbers and are susceptible to direct and indirect ultraviolet damage. The previous site-directed mutagenesis studies have revealed a striking difference for two highly conserved homologous ?-hairpin Tyr pairs, at the N-terminal domain (N-td) and C-terminal domain (C-td), respectively, in their contribution to the overall stability of H?D-Crys, but why they behave so differently still remains a mystery. In this paper, we systematically investigated the underlying molecular mechanism and detailed contributions of these two Tyr pairs with large scale molecular dynamics simulations. A series of different tyrosine-to-alanine pair(s) substitutions were performed in either the N-td, the C-td, or both. Our results suggest that the Y45A/Y50A pair substitution in the N-td mainly affects the stability of the N-td itself, while the Y133A/Y138A pair substitution in the C-td leads to a more cooperative unfolding of both N-td and C-td. The stability of motif 2 in the N-td is mainly determined by the interdomain interface, while motif 1 in the N-td or motifs 3 and 4 in the C-td are mainly stabilized by the intradomain hydrophobic core. The damage to any tyrosine pair(s) can directly introduce some apparent water leakage to the hydrophobic core at the interface, which in turn causes a serious loss in the stability of the N-td. However, for the C-td substitutions, it may further impair the stable "sandwich-like" Y133-R167-Y138 cluster (through cation-? interactions) in the wild-type, thus causing the loop regions near the residue A138 to undergo large fluctuations, which in turn results in the intrusion of water into the hydrophobic core of the C-td and induces the C-td to lose its stability. These findings help resolve the "mystery" on why these two Tyr pairs display such a striking difference in their contributions to the overall protein stability despite their highly homologous nature.

Project description:Cataract, a major cause of visual impairment worldwide, is the opacification of the eye's crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

Dataset Information

Contributions of aromatic pairs to the folding and stability of long-lived human ?D-crystallin.

Publications

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

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