Beta A3/A1-crystallin association: role of the N-terminal arm.
The beta- and gamma-crystallins of the lens form a protein superfamily, the beta gamma-crystallins and have highly conserved two-domain core structures. Whereas gamma-crystallins exist as monomers, the beta-crystallins associate into large aggregates. The N-terminal extensions to the core domains of beta-crystallins are postulated to be essential for their aggregation characteristics. To test this hypothesis, we compared the aggregation properties of a recombinant mouse beta A3/A1-crystallin without its N-terminal extension (r beta A3tr) to a normal recombinant mouse beta A3/A1-crystallin (r beta A3). The identity of the baculo-virus system-expressed recombinant crystallins was confirmed by gel electrophoresis, immunoblots and N-terminal sequence analysis. Circular dichroism measurements indicate that the recombinant crystallins have mostly beta-sheet conformation, similar to normal beta-crystallins. The normal r beta A3 migrates on gel filtration chromatography as a homodimer, whereas the r beta A3tr migrates mostly as a monomer. After relocating the recombinant crystallins with mouse lens soluble extract, r beta A3 migrated with the dimeric beta L2 fractions and to a lesser extent with tetrameric beta L1 fractions. The reassociated r beta A3tr migrated with the trailing edge of the beta L 2 fractions (40 kDa). These results suggest that the N-terminal arm of beta A3/A1-crystallin facilitates dimer formation and is necessary for higher-order associations.
Hope
J N
JN
Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, MD 20892.
Chen
H C
HC
Hejtmancik
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D015151:Immunoblotting; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
D003459:Crystallins; D046911:Macromolecular Substances; D011994:Recombinant Proteins