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Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment.

ABSTRACT: The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.

SUBMITTER: MacDonald JT

PROVIDER: S-EPMC2253261 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment.

MacDonald James T JT Purkiss Andrew G AG Smith Myron A MA Evans Paul P Goodfellow Julia M JM Slingsby Christine C

Protein science : a publication of the Protein Society 20050501 5

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular d ...[more]

PMID: 15840832

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Project description:In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the beta/gamma-crystallin family share common beta-sheet rich domains and hydrophobic regions at the monomer-monomer or domain-domain interfaces. Disruption of these interfaces, due to post-translational modifications, such as deamidation, decreases the stability of the crystallins. Previous experiments have failed to define the structural changes associated with this decreased stability. Using hydrogen/deuterium exchange with mass spectrometry (HDMS), deamidation-induced local structural changes in betaB2-crystallin were identified. Deamidation was mimicked by replacing glutamines with glutamic acids at homologous residues 70 and 162 in the monomer-monomer interface of the betaB2-crystallin dimer. The exchange-in of deuterium was determined from 15 s to 24 h and the global and local changes in solvent accessibility were measured. In the wild type betaB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of betaB2-crystallin in solution. This is consistent with a tightly packed domain structure observed in the crystal structure. Deuterium levels were initially greater in N-terminal domain (N-td) peptides than in homologous peptides in the C-terminal domain (C-td). The more rapid incorporation suggests a greater solvent accessibility of the N-td. In the betaB2-crystallin crystal structure, interface Gln are oriented towards their opposite domain. When deamidation was mimicked at Gln70 in the N-td, deuterium levels increased at the interface peptide in the C-td. A similar effect in the N-td was not observed when deamidation was mimicked at the homologous residue, Gln162, in the C-td. This difference in the mutants can be explained by deamidation at Gln70 disrupting the more compact C-td and increasing the solvent accessibility in the C-td interface peptides. When deamidation was mimicked at both interface Gln, deuterium incorporation increased in the C-td, similar to deamidation at Gln70 alone. In addition, deuterium incorporation was decreased in the N-td in an outside loop peptide adjacent to the mutation site. This decreased accessibility may be due to newly exposed charge groups facilitating ionic interactions or to peptides becoming more buried when other regions became more exposed. The highly sensitive HDMS methods used here detected local structural changes in solution that had not been previously identified and provide a mechanism for the associated decrease in stability due to deamidation. Changes in accessibility due to deamidation at the interface led to structural perturbations elsewhere in the protein. The cumulative effects of multiple deamidation sites perturbing the structure both locally and distant from the site of deamidation may contribute to aggregation and precipitation during aging and cataractogenesis in the lens.

Project description:Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the ?- and ?-crystallins. Here, we investigated the effects of the deamidation of ?A- and ?B-crystallins on their interaction with ?A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT ?A- and WT ?B-crystallins and their deamidated mutants with ?A3-crystallin. The deamidated mutants of ?A-crystallin (?A N101D and ?A N123D) displayed lower adherence propensity for ?A3-crystallin relative to the binding affinity shown by WT ?A-crystallin. Among ?B-crystallin mutants, ?B N78D displayed higher adherence propensity whereas ?B N146D mutant showed slightly lower binding affinity for ?A3-crystallin relative to that shown by WT ?B-crystallin. Under the in vivo condition (FLIM-FRET), both ?A-deamidated mutants (?A N101D and ?A N123D) exhibited strong interaction with ?A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT ?A-crystallin (18±4%). Similarly, the ?B N78D and ?B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ?A3-crystallin compared to 18±4% FRET efficiency of WT ?B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of ?A- and ?B-crystallins with ?A3-crystallin suggested that interaction sites most likely reside in the ?A CTE and ?B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT ?A- and WT?B-crystallins, the deamidated mutants showed strong interactionfor ?A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of ?-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Dataset Information

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment.

Publications

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment.

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