Solvent accessibility of βB2-crystallin and local structural changes due to deamidation at the dimer interface

Takumi Takata, Joshua P. Smith, Brian Arbogast, Larry David, Kirsten Lampi

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the β/γ-crystallin family share common β-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 βB2-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 βB2-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 βB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of βB2-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 βB2-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.

Original languageEnglish (US)
Pages (from-to)336-346
Number of pages11
JournalExperimental Eye Research
Volume91
Issue number3
DOIs
StatePublished - Sep 2010

Fingerprint

Crystallins
Deuterium
Hydrogen
Lenses
Mass Spectrometry
Glutamates
Peptides
Crystalline Lens
Post Translational Protein Processing
Glutamine
Amides
Proteins
Mutation

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Solvent accessibility of βB2-crystallin and local structural changes due to deamidation at the dimer interface. / Takata, Takumi; Smith, Joshua P.; Arbogast, Brian; David, Larry; Lampi, Kirsten.

In: Experimental Eye Research, Vol. 91, No. 3, 09.2010, p. 336-346.

Research output: Contribution to journalArticle

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abstract = "In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the β/γ-crystallin family share common β-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 βB2-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 βB2-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 βB2-crystallin (WT), only about 20{\%} of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of βB2-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 βB2-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.",
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N2 - In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the β/γ-crystallin family share common β-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 βB2-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 βB2-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 βB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of βB2-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 βB2-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.

AB - In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the β/γ-crystallin family share common β-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 βB2-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 βB2-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 βB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of βB2-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 βB2-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.

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