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

Protein Sci. 2007 Nov;16(11):2427-44. doi: 10.1110/ps.072970207. Epub 2007 Sep 28.


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 gammaD-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 gammaD-crystallin, as well as the isolated domains of human gammaS-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 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 gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Crystallins / chemistry*
  • Escherichia coli / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lens, Crystalline / metabolism*
  • Models, Molecular
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Spectrometry, Fluorescence / methods
  • Temperature
  • gamma-Crystallins / chemistry*


  • CRYGD protein, human
  • Crystallins
  • gamma-Crystallins
  • CRYGS protein, human