The C-terminal domains of gammaS-crystallin pair about a distorted twofold axis

The 2-domain gammaS-crystallin, a highly conserved early evolutionary off-shoot of the gamma-crystallin family, is located in the water-rich region of eye lenses. The expressed C-terminal domain, gammaS-C, has been crystallized and the 2.56 A X-ray structure determined. There are two domains in the asymmetric unit which pair about a distorted twofold axis. One of the domains has an altered conformation in a highly conserved region of the protein, the tyrosine corner. The distorted gammaS-C dimer of domains is compared with the highly symmetrical, equivalent recombinant dimer of C-terminal domains from gammaB- crystallin. Sequence changes close to the interface, that distinguish gammaS from the other gamma-crystallins, are examined in order to evaluate their role in symmetrical domain pairing.      

The role of the sequence extensions in {beta}-crystallin assembly

The modular construction of the eye lens ß-crystallinsmakes them good candidates for protein engineering to ascertainthe rules of assembly of oligomers. X-ray studies have shownthat although the polypeptide chains of ßB2-crystallinand -crystallins fold to form similar N- and C-terminal domains,the conformation of the connecting peptides are such that the-crystallins are monomers and the ß-crystallin isa dimer. Unlike -crystallins, the numerous -crystallins haveextensions of variable sequence from the globular domains. Wehave tested the effect of removing the N- and C-terminal extensionsfrom rat ßB2-crystallin using a bacterial expressionsystem. Abundant proteins were produced in Escherichia coliusing the pET or pQE vectors. Full-length and truncated proteinswere purified and checked for refolding using circular dichroism.Sizing of the truncated proteins using gel filtration chroma-tographyshowed that the absence of either the N- or C-terminal extensiondoes not affect dimerization of ßB2-crystallin.