Folding and stability of the isolated Greek key domains of the long-lived human lens proteins γD-crystallin and γS-crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gamma D-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N-and C-terminal domains of human gD-crystallin, as well as the isolated domains of human gamma Scrystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/ refolding experiments. The decrease in stability of the N-terminal domain of human gamma D-crystallin with respect to the complete protein indicated that the interdomain interface contributes Delta G(H2O) of 4.2 kcal/ mol to the overall stability of this very long-lived protein.