/3A3/Al-crystallin association: role of the N-terminal arm John N.Hope1, Hao-Chia Chen2

^ o whom correspondence should be addressed The /3- and 7-crystallins of the lens form a protein super-family, the /37-crystallins and have highly conserved two-domain core structures. Whereas 7-crystallins exist as monomers, the /3-crystallins associate into large aggregates. The N-terminal extensions to the core domains of /3-crystallins are postulated to be essential for their aggregation charac-teristics. To test this hypothesis, we compared the aggregation properties of a recombinant mouse /3A3/Al-crj stallin without its N-terminal extension (r/3A3tr) to a normal recombinant mouse /SA3/Al-crystallin (r/SA3). The identity of the baculo-virus system-expressed recombinant crystallins was confirmed by gel ekctrophoresis, immunoblots and N-terminal sequence analysis. Circular dichroism measurements indicate that the recombinant crystallins have mostly /3-sheet conformation, similar to normal /3-crystallins. The normal r/3A3 migrates on gel filtration chromatography as a homodimer, whereas the r/3A3tr migrates mostly as a monomer. After relocating the recombinant crystallins with mouse lens soluble extract, r/3A3 migrated with the dimeric /3L2 fractions and to a lesser extent with tetrameric /3L1 fractions. The reassociated r/3A3tr migrated with the trailing edge of the /3L2 fractions (40 kDa). These results suggest that the N-terminal arm of /3A3/A1-crystaUin facUitates dimer formation and is necessary for higher-order associations. Key words: baculovirus expression system/lens protein reassocia-tion/m vitro mutagenesi