The folding landscape of an a-lytic protease variant reveals the role of a conserved b-hairpin in the development of kinetic stability

Most secreted bacterial proteases, including a-lytic protease (aLP), are synthesized with covalently attached pro regions necessary for their folding. The aLP folding landscape revealed that its pro region, a potent folding catalyst, is required to circumvent an extremely large folding free energy of activation that appears to be a consequence of its unique unfolding transition. Remarkably, the aLP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although aLP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved b-hairpin in the C-terminal domain was identified as a structural element whose formation and positioning may contribute to the large folding free energy barrier. In this article, we characterize the folding of an aLP variant with a more favorable b- hairpin turn conformation (aLP sub(b-turn)). Indeed, aLP sub(b-turn) pro region-catalyzed folding is faster than that for aLP. However, instead of accelerating spontaneous folding, aLP sub(b-turn) actually unfolds more slowly than aLP. Our data support a model where the b-hairpin is formed early, but its packing with a loop in the N-terminal domain happens late in the folding reaction. This tight packing at the domain interface enhances the kinetic stability of aLP sub(b-turn), to nearly the same degree as the change between aLP and a faster folding homolog. However, aLP sub(b-turn) has impaired proteolytic activity that negates the beneficial folding properties of this variant. This study demonstrates the evolutionary limitations imposed by the simultaneous optimization of folding and functional properties. Proteins 2005.