Acetylation of alpha-cryatallin with N-acetylimidazole and its influence upon the native aggregate and subunit reassembly

Purpose; An attempt has been made to investigate the involvement and import ance of some of the hydrogen bond forming amino acid side chains in intra a nd inter subunit interactions in alpha-crystallin assembly. Methods. For this, alpha-crystallin has been acetylated, partially or compl etely, using N-acetylimidazole, The apparent molecular size, electrophoreti c mobility, conformational properties, surface hydrophobicity and chaperone activity of the modified proteins have been determined and compared with t hose of unmodified native protein as well as of the aggregates reassembled from the modified subunits. Results. Acetylation of the surface-exposed tyrosine side chains has been f ound to destabilize the integrity of the native assembly with the formation of a somewhat smaller aggregate. This acetylated aggregate appears to adop t a molten globule-like conformation as evidenced from its almost unaltered secondary structure with some detectable alterations in its tertiary struc ture as well as from its enhanced chaper-one activity exhibited by the redu ction assay compared to the native alpha-crystallin. Reassociation Studies from either partially or completely acetylated subunits indicate that acety lation perturbs the information needed for native refolding of the subunits from their unfolded state as well as that needed for the normal mode of su bunit reassembly. Acetylated subunits exhibit abnormal gel electrophoretic band pattern with distinctly retarded migration compared to the unmodified subunits. However, in spite of the partial/complete acetylation of the subu nits or their reassociation from the denatured state, the tryptophan fluore scence emission maxima of the modified proteins and also that of the reasso ciated aggregates appear to remain unaffected. Conclusions. Results tend to indicate that the unperturbed hydrogen bonding capability of the relevant side chains in alpha-crystallin is needed for t he integrity of the native alpha-crystallin assembly, for the normal refold ing of its denatured subunits and also for the correct mode of subunit reas sembly.