Aggregation of beta A3-crystallin is independent of the specific sequence of the domain connecting peptide.

The beta- and gamma-crystallins are structural proteins whose high concentration and close packing are important in maintaining transparency of the eye lens. The beta gamma-crystallin superfamily includes proteins with similar core structures consisting of two compact domains linked by a short connecting peptide. In gamma-crystallins, the connecting peptide folds back on itself, allowing the amino and carboxyl domains to participate in close interactions. The beta-crystallin connecting peptide is extended so that dimerization of two beta-crystallin monomers is required for similar interdomain interactions. In order to examine the role of the sequence of the connecting peptide in determining the extended beta-crystallin conformation and hence their association into dimers, we have exchanged the 10 residues of the beta A3-crystallin connecting peptide with the 9-residue connecting peptide sequence of mouse gamma B-crystallin by site-directed mutagenesis. Unaltered and modified recombinant beta A3-crystallins were expressed in a baculovirus system and purified by sequential anion exchange chromatography and gel filtration. Integrity of the recombinant crystallins was confirmed by NH2-terminal sequence analysis, immunoblots, and CD spectrometry. Reconstitution of the mutant recombinant protein with crystallins from mouse lens soluble extract resulted in aggregates of identical size distribution as normal beta A3-crystallin. We conclude that the sequence of the connecting peptide is not critical for the association of beta A3-crystallin into dimers and higher order aggregates as had been postulated.