Interdomain side-chain interactions in human gammaD crystallin influencing folding and stability

Protein Sci. 2005 Aug;14(8):2030-43. doi: 10.1110/ps.051460505.


Human gammaD crystallin (HgammaD-Crys) is a two domain, beta-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HgammaD-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among gamma-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HgammaD-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37 degrees C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of HgammaD-Crys that could be significant in cataract.

Publication types

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

MeSH terms

  • Amino Acid Substitution
  • Amino Acids / chemistry
  • Arginine / chemistry
  • Arginine / genetics
  • Circular Dichroism
  • Crystallins / chemistry*
  • Crystallins / genetics
  • Crystallins / metabolism
  • Glutamine / chemistry
  • Glutamine / genetics
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Methionine / chemistry
  • Methionine / genetics
  • Models, Molecular
  • Protein Folding
  • Protein Structure, Tertiary
  • Spectrometry, Fluorescence
  • gamma-Crystallins / chemistry*
  • gamma-Crystallins / genetics
  • gamma-Crystallins / metabolism


  • Amino Acids
  • CRYGD protein, human
  • Crystallins
  • gamma-Crystallins
  • Glutamine
  • Arginine
  • Methionine