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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.
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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).
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.
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.
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.
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