April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Amide Hydrogen Exchange Study for Local Structural Alteration in βA3-crystallin
Author Affiliations & Notes
  • T. Takata
    Integrative Biosci, Oregon Health & Sciences Univ, Portland, Oregon
  • K. Lampi
    Integrative Biosci, Oregon Health & Sciences Univ, Portland, Oregon
  • Footnotes
    Commercial Relationships  T. Takata, None; K. Lampi, None.
  • Footnotes
    Support  NIH NEI EY 012239
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1639. doi:
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      T. Takata, K. Lampi; Amide Hydrogen Exchange Study for Local Structural Alteration in βA3-crystallin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1639.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : In higher-ordered lens protein complexes, β-crystallin subunits have common domain structures, and a hydrophobic interactions at the monomer-monomer or domain-domain interface. During aging, the of conserved glutamine residues at the interface undergo deamidation. Deamidation is the most extensive post-translational modification during aging (Wilmarth et al, 2006). The purpose of this study was to investigate deamidation on local structural changes in this interface using by hydrogen/deuterium exchange with mass spectrometry (HXMS).

Methods: : Two sites of deamidation were mimicked in βA3 by replacing the glutamines with glutamic acids by site-directed mutagenesis (DM). The dimer formation of WT βA3 or DM βA3 size exclusion chromatography and multiangle laser light scattering, H/D-exchange coupled with ESI-Q-Tof mass spectrometry (HXMS) was performed. Hydrogen exchange of amide backbone βA3 was started by 10-fold dilution in deuterium oxide. At each time, a small amount of protein was aliquoted and back exchange was quenched from deuterium to hydrogen by immediately freezing. For protein "global" level analysis, sample was injected ESI-Q-tof mass spectrometry under back exchange quenching condition. For peptide "local" level analysis of interface, each sample was digested by pepsin with GuHCl before injection into mass spectrometry.

Results: : In global HXMS, deamidation did not change deuterated ratio in βA3, suggesting deamidation did not change βA3 total conformation. Interestingly, HXMS at the peptide level did indicate differences between WT and DM, suggesting deamidation at the intrface induced a space between each subunit or domain structures. HXMS results fit well for secondary structure prediction and modeled structure and additionally localize the structural differences by differences in H/D dynamics at the peptide level.

Conclusions: : The HXMS detected by mass spectrometry is an extremely valuable method for understanding crystallin structure and crystallin-crystallin interactions. These results from HXMS for βA3-crystallin suggests that deamidation induced a cavity in β-sheet strand at the hydrophobic interface.

Keywords: crystallins • protein modifications-post translational 

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