Characterization of a human α1-antitrypsin variant that is as stable as ovalbumin

The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central beta-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase, We have examined the folding and inhibitory activity of a very stable variant of human alpha(1)-antitrypsin, a prototype inhibitory serpin, A combination of seven stabilizing single amino acid substitutions of alpha(1)-antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin, Compared with the wild-type alpha(1)-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state, Surprisingly, Multi-7 alpha(1)-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A sheet closure conferred by Multi-7 does not affect the complex formation, It may be that the stability increase of Multi-7 alpha(1)-antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.