J K Pal1, S K Bera, S K Ghosh. 1. Crystallography & Molecular Biology Division, Saha Institute of Nuclear Physics 1/AF Bidhannagar, 700064, Calcutta, India.
Abstract
PURPOSE: An attempt has been made to investigate the involvement and importance of some of the hydrogen bond forming amino acid side chains in intra and inter subunit interactions in alpha-crystallin assembly. METHODS: For this, alpha-crystallin has been acetylated, partially or completely, using N-acetylimidazole. The apparent molecular size, electrophoretic mobility, conformational properties, surface hydrophobicity and chaperone activity of the modified proteins have been determined and compared with those 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 found to destabilize the integrity of the native assembly with the formation of a somewhat smaller aggregate. This acetylated aggregate appears to adopt a molten globule-like conformation as evidenced from its almost unaltered secondary structure with some detectable alterations in its tertiary structure as well as from its enhanced chaper-one activity exhibited by the reduction assay compared to the native alpha-crystallin. Reassociation studies from either partially or completely acetylated subunits indicate that acetylation perturbs the information needed for native refolding of the subunits from their unfolded state as well as that needed for the normal mode of subunit 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 subunits or their reassociation from the denatured state, the tryptophan fluorescence emission maxima of the modified proteins and also that of the reassociated 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 the integrity of the native alpha-crystallin assembly, for the normal refolding of its denatured subunits and also for the correct mode of subunit reassembly.
PURPOSE: An attempt has been made to investigate the involvement and importance of some of the hydrogen bond forming amino acid side chains in intra and inter subunit interactions in alpha-crystallin assembly. METHODS: For this, alpha-crystallin has been acetylated, partially or completely, using N-acetylimidazole. The apparent molecular size, electrophoretic mobility, conformational properties, surface hydrophobicity and chaperone activity of the modified proteins have been determined and compared with those 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 found to destabilize the integrity of the native assembly with the formation of a somewhat smaller aggregate. This acetylated aggregate appears to adopt a molten globule-like conformation as evidenced from its almost unaltered secondary structure with some detectable alterations in its tertiary structure as well as from its enhanced chaper-one activity exhibited by the reduction assay compared to the native alpha-crystallin. Reassociation studies from either partially or completely acetylated subunits indicate that acetylation perturbs the information needed for native refolding of the subunits from their unfolded state as well as that needed for the normal mode of subunit 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 subunits or their reassociation from the denatured state, the tryptophan fluorescence emission maxima of the modified proteins and also that of the reassociated 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 the integrity of the native alpha-crystallin assembly, for the normal refolding of its denatured subunits and also for the correct mode of subunit reassembly.