Distribution of the native strain in human alpha(1)-antitrypsin and its association with protease inhibitor function

Serine protease inhibitors (serpins) are metastable in their native state. This strain, which is released upon binding to target proteases, is essenti al for the inhibitory activity of serpins. To understand the structural bas is of the native strain, we previously characterized stabilizing mutations of alpha(1)-antitrypsin, a prototypical inhibitory serpin, in regions such as the hydrophobic core. The present study evaluates the effects of single point mutations throughout the molecule on stability and protease inhibitor y activity. We identified stabilizing mutations in most secondary structure s, suggesting that the native strain is distributed throughout the molecule . Examination of the substitution patterns and the structures of the mutati on sites revealed surface hydrophobic pockets as a component of the native strain in alpha(1)-antitrypsin, in addition to the previously identified un usual interactions such as side chain overpacking and cavities. Interesting ly, many of the stabilizing substitutions did not affect the inhibitory act ivity significantly. Those that affected the activity were confined in the regions that are mobilized during the complex formation with a target enzym e. The results of our study should be useful for designing proteins with st rain and for regulating the stability and functions of serpins.