Domain exchange experiments in duck delta-crystallins: Functional and evolutionary implications

delta-Crystallin, the major soluble protein component of the avian and rept ilian eye lens, is homologous to the urea cycle enzyme argininosuccinate ly ase (ASL). In duck lenses there are two delta crystallins, denoted delta 1 and delta 2. Duck delta 2 is both a major structural protein of the lens an d also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta 2/ASL in the amino acid sequence, delta 1 is enzyma tically inactive. A series of hybrid proteins have been constructed to asse ss the role of each structural domain in the enzymatic mechanism. Five chim eras-221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta 1 or delta 2, respectively-were constructed and thermodynami cally and kinetically analyzed. The kinetic analysis indicates that only do main 1 is crucial for restoring ASL activity to delta 1 crystallin, and tha t amino acid substitutions in domain 2 may play a role in substrate binding . These results confirm the hypothesis that only one domain, domain I, is r esponsible for the loss of catalytic activity in delta 1. The thermodynamic characterization of human ASL (hASL) and duck delta 1 and delta 2 indicate that delta crystallins are slightly less stable than hASL, with the delta 1 being the least stable. The Delta Gs of unfolding are 57.25, 63.13, and 7 0.71 kcal mol(-1) for delta 1, delta 2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to th eir structural role by adopting a slightly less stable conformation that mi ght allow for enhanced protein-protein and protein-solvent interactions.