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