Molecular evolution of an extremely stable PAI-1 variant

Earlier studies have demonstrated that the cooperative effect of different mutations are required to increase stability of plasminogen activator inhibitor 1 (PAI-1).1 Therefore, we used a PAI-1 mutant library containing an average of 9.1 amino acid substitutions per protein to screen and select for variants with a prolonged half-life of the active conformation. Screening was performed by incubating PAI-1-presenting phages for increasing periods of time (16-100 h) at 37°C, followed by stringent selection for active PAI-1 variants that bind tissue-type plasminogen activator (t-PA). After selection of the most stable variant, the contributions of the individual mutations were assessed by DNA shuffling of this mutant with an excess of wild-type PAI-1 cDNA (i.e. back-crossing). Screening of the original PAI-1 mutant library resulted in 7 clones with prolonged halflives of which the most stable variant showed a 140-fold increase as compared to wild-type (345 vs. 2.5 h). This mutant contains 11 amino acid substitutions, does not display substrate behavior, and has a second order rate constant of inhibition that is only 2 to 3-fold lower than that of wildtype PAI-1, Back-crossing of this mutant, and subsequent selection, revealed that a minimum of 7 amino acid substitutions is necessary to substantially increase the stability of PAI-1 without affecting inhibitory vs. substrate behavior. The mutations are found distant from each other, suggesting a general impact on the structure of PAI-1 and stressing the need for co-operative mutations to increase PAI-1 stability.