The Folding Landscape of an -Lytic Protease Variant Reveals the Role of a Conserved -Hairpin in the Development of Kinetic Stability

Most secreted bacterial proteases, including -lytic protease ( LP), are synthesized with covalently attached pro regions necessary for their folding. The LP 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 LP native state is thermodynamically unstable; a large unfolding free energy barrier is solely responsible for the persistence of its native state. Although LP folding is well characterized, the structural origins of its remarkable folding mechanism remain unclear. A conserved -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 LP variantwith amore favorable -hairpin turn conformation ( LP -turn). Indeed, LP -turn pro regioncatalyzed folding is faster than that for LP. However, instead of accelerating spontaneous folding, LP -turn actually unfolds more slowly than LP. Our data support a model where the -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 LP -turn, to nearly the same degree as the change between LP and a faster folding homolog. However, LP -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;61:105–114. © 2005Wiley-Liss, Inc.