AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin

Biochemistry. 2004 Dec 21;43(50):15785-95. doi: 10.1021/bi048151s.


Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Cattle
  • Circular Dichroism
  • Heat-Shock Proteins / chemistry*
  • Heat-Shock Proteins / metabolism
  • Humans
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / metabolism
  • Molecular Sequence Data
  • Mutation / genetics
  • Peptide Library
  • Peptide Mapping / methods
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism
  • Protein Interaction Mapping
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Sequence Analysis, Protein
  • alpha-Crystallin A Chain / chemistry*
  • alpha-Crystallin A Chain / metabolism
  • alpha-Crystallin B Chain / chemistry*
  • alpha-Crystallin B Chain / genetics
  • alpha-Crystallin B Chain / metabolism


  • Heat-Shock Proteins
  • Molecular Chaperones
  • Peptide Library
  • Peptides
  • Protein Subunits
  • alpha-Crystallin A Chain
  • alpha-Crystallin B Chain