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Solution properties of ?-crystallins: compact structure and low frictional ratio are conserved properties of diverse ?-crystallins.

ABSTRACT: ?-crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, ?-crystallins have high contents of sulfur-containing methionine and cysteine, but this reaches extremes in fish ?M-crystallins with up to 15% Met. In addition, fish ?M-crystallins do not conserve the paired tryptophan residues found in each domain in mammalian ?-crystallins and in the related ?-crystallins. To gain insight into important, evolutionarily conserved properties and functionality of ?-crystallins, zebrafish (Danio rerio) ?M2b and ?M7 were compared with mouse ?S and human ?D. For all four proteins, far UV CD spectra showed the expected ?-sheet secondary structure. Like the mammalian proteins, ?M7 was highly soluble but ?M2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full-length and truncated versions of human ?B-crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse ?-crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of ?-crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.

SUBMITTER: Chen Y

PROVIDER: S-EPMC3892301 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Project description:As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.

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Solution properties of ?-crystallins: compact structure and low frictional ratio are conserved properties of diverse ?-crystallins.

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