Phe71 is essential for chaperone-like function in alpha A-crystallin

J Biol Chem. 2001 Dec 14;276(50):47094-9. doi: 10.1074/jbc.M107737200. Epub 2001 Oct 11.


Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Anilino Naphthalenesulfonates / pharmacology
  • Animals
  • Circular Dichroism
  • Citrate (si)-Synthase / chemistry
  • Cloning, Molecular
  • Crystallins / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Fluorescent Dyes / pharmacology
  • Insulin / metabolism
  • Molecular Chaperones / chemistry
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phenylalanine / chemistry*
  • Phenylalanine / physiology*
  • Protein Binding
  • Protein Denaturation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence
  • Temperature
  • Time Factors
  • Tryptophan / chemistry
  • Ultraviolet Rays


  • Anilino Naphthalenesulfonates
  • Crystallins
  • Fluorescent Dyes
  • Insulin
  • Molecular Chaperones
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Phenylalanine
  • 5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)
  • Tryptophan
  • Citrate (si)-Synthase