May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Association Properties of βB1- and βA3-Crystallins: Ability to Form Heterotetramers
Author Affiliations & Notes
  • M. B. Dolinska
    OGVFB, NEI/NIH, Bethesda, Maryland
  • M. P. Chan
    OGVFB, NEI/NIH, Bethesda, Maryland
  • Y. V. Sergeev
    OGVFB, NEI/NIH, Bethesda, Maryland
  • P. T. Wingfield
    NIAMS/NIH, Bethesda, Maryland
  • F. J. Hejtmancik
    OGVFB, NEI/NIH, Bethesda, Maryland
  • Footnotes
    Commercial Relationships  M.B. Dolinska, None; M.P. Chan, None; Y.V. Sergeev, None; P.T. Wingfield, None; F.J. Hejtmancik, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4099. doi:https://doi.org/
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      M. B. Dolinska, M. P. Chan, Y. V. Sergeev, P. T. Wingfield, F. J. Hejtmancik; Association Properties of βB1- and βA3-Crystallins: Ability to Form Heterotetramers. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4099. doi: https://doi.org/.

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

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Abstract

Purpose: : Crystallins are the major water-soluble highly concentrated structural proteins of the lens where there are responsible for the transparency and high refractive index. The most heterogeneous β-crystallins form aggregates of different sizes and associate into dimers, tetramers, and higher-order complexes with other β-crystallins. In order to enhance our understanding of the interactions between β-crystallins, this study characterizes the association of βB1-crystallin (βB1), a major component of large β-crystallin complexes (β-high) with itself and with βA3-crystallin (βA3).

Methods: : Both crystallins were expressed in E.coli and purified from the soluble fraction using ion-exchange and size-exclusion chromatography. Their association was then characterized using Superdex 75 analytical grade column, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation.

Results: : When present alone, each protein associates into homodimers but neither forms homotetramers. Upon mixing under physiological conditions, heterocomplex formation between βB1 and βA3 was observed by size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to the previous results obtained from βB2 and βA3, which did not indicate tetramer formation, analytical centrifugation shows a dimer-tetramer equilibrium with a Kd of 1.1×10-6 µM, suggesting that βB1 and βA3 associate predominantly into heterotetramers in vitro.

Conclusions: : While each purified β-crystallin associates only into homodimers and mixed βB2 and βA3 form a mixture of homo- and heterodimers, mixed βB1/βA3 associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for βB1 in promoting higher-order crystallin association in the lens.

Keywords: crystallins • protein structure/function • protein purification and characterization 
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