QUATERNARY STRUCTURE OF BOVINE ALPHA-CRYSTALLIN - SURFACE PROBING BY LIMITED PROTEOLYSIS INVITRO

Limited proteolysis of α‐crystallin with 10 different endopeptidases in vitro shows that only 5–20 residues of the carboxy termini of A and B chains are accessible to these enzymes, without disruption of the quaternary structure. The A chains are more susceptible to proteolysis in vitro than B chains, which corresponds with the degradation in vivo upon aging of α‐crystallin in the lens. Six proteases specifically cleave off the carboxyl‐terminal pentnpcptide of all B chains, which indicates that the B (Thr170 ‐Ala171) bond, the only observed degradation site of B chains in vivo, is surface‐exposed and proteolytically very labile. None of the endopeptidases split the A (Asp151 ‐Ala152) bond, the main degradation site of A chains in vivo, but a nearby segment A156–163 iS very accessible to several enzymes. We postulate that this A151–152 bond is close to the surface of α‐crystallin and, although proteolytically stable in the native protein, it could become exposed as a result of age‐related changes in secondary and/or tertiary structure. Two minor tryptic cleavage sites in A chains of native α‐crystallin become more accessible in reassociated a‐crystallin, and particularly in pure A‐chain reaggregates, which indicates a change in secondary/tertiary structure upon dissociation‐reassociation. Although the accessibility of B chains suggest that they are in equivalent positions in the α‐crystallin quaternary structure, the A chains exhibit various rates of proteolysis and therefore occur in more than one orientation. The anomalously slow migration of B chain in dodecylsulfate gel electrophoresis is abolished after removal of its carboxyl‐terminal pentapeptide, Ala‐Ala‐Pro‐Lys‐Lys. Copyright © 1979, Wiley Blackwell. All rights reserved