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

¹ Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
² Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa 50011, USA
³ Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA

(RECEIVED April 27, 2007; FINAL REVISION July 23, 2007; ACCEPTED August 1, 2007)

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric -crystallins and oligomeric -crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human 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 D-crystallin, as well as the isolated domains of human S-crystallin. 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°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 D-crystallin with respect to the complete protein indicated that the interdomain interface contributes G _H2O of 4.2 kcal/mol to the overall stability of this very long-lived protein.

Keywords: human D-crystallin; human S-crystallin; domain interface; cataract; equilibrium unfolding/refolding transitions; refolding kinetic intermediates; protein stability

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072970207.

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