Eye lens betaB2-crystallin: circular permutation does not influence the oligomerization state but enhances the conformational stability.

The related vertebrate eye lens polypeptides, betaB2- and gammaB-crystallin, each fold into two similar beta-sheet domains. The main difference is the state of oligomerization resulting from intermolecular domain interactions in the oligomeric beta-crystallins and intramolecular contacts in the monomeric gamma-crystallins. The question arises whether it is possible to create a monomeric gammaB-like betaB2-molecule by protein engineering methods. We wanted to produce such a molecule by circularly permuting the domains of betaB2-crystallin. The new termini were created from the original connecting peptide, and the new linker from stumps of the original extensions, while the rest of the flexible extensions were deleted. As judged by circular dichroism and fluorescence, the permutation causes little change in the structure of the protein. The circularly permuted protein forms dimers as wild-type betaB2-crystallin. On the other hand, cpbetaB2 shows a slightly enhanced stability against urea with a midpoint of transition of 2.1 M urea versus 1.9 M for the wild-type protein lacking N and C-terminal arms, thus indicating stronger domain interactions. To our knowledge this is the first circularly permuted protein which exhibits a higher stability than the corresponding wild-type protein.