alpha-Lytic protease (alphaLP), an extracellular bacterial protease, is synthesized with a large amino-terminal pro-region that is essential for its folding in vivo and in vitro. In the absence of the pro-region, the protease folds to an inactive, partially folded state, designated 'I'. The pro-region catalyses protease folding by directly stabilizing the folding transition state (>26kcal mol(-1)) which separates the native state 'N' from I. Although a basic tenet of protein folding is that the native state of a protein is at the minimum free energy, we show here that both the I and fully unfolded states of alphaLP are lower in free energy than the native state. Native alphaLP is thus metastable: its apparent stability derives from a large barrier to unfolding. Consequently, the evolution of alphaLP has been distinct from most other proteins: it has not been constrained by the free-energy difference between the native and unfolded states, but instead by the size of its unfolding barrier.

During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial alpha-lytic protease (alphaLP) these two processes have become decoupled. The native state of alphaLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 approximately 1,800 years) to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving alphaLP trapped in its native state by a large kinetic unfolding barrier (t1/2 approximately 1.2 years). alphaLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of alphaLP by the optimal suppression of proteolytic sensitivity.

The folding of the extracellular serine protease, ␣-lytic protease (␣LP; EC 3.4.21.12) reveals a novel mechanism for stability that appears to lead to a longer functional lifetime for the protease. For ␣LP, stability is based not on thermodynamics, but on kinetics. Whereas this has required the coevolution of a pro region to facilitate folding, the result has been the optimization of native-state properties independent of their consequences on thermodynamic stability. Structural and mutational data lead to a model for catalysis of folding in which the pro region binds to a conserved ␤-hairpin in the ␣LP C-terminal domain, stabilizing the folding transition state and the native state. The pro region is then proteolytically degraded, leaving the active ␣LP trapped in a metastable conformation. This metastability appears to be a consequence of pressure to evolve properties of the native state, including a large, highly cooperative barrier to unfolding, and extreme rigidity, that reduce susceptibility to proteolytic degradation. In a test of survival under highly proteolytic conditions, homologous mammalian proteases that have not evolved kinetic stability are much more rapidly degraded than ␣LP. Kinetic stability as a means to longevity is likely to be a mechanism conserved among the majority of extracellular bacterial pro-proteases and may emerge as a general strategy for intracellular eukaryotic proteases subject to harsh conditions as well.Virtually all extracellular bacterial proteases are synthesized as precursor molecules with pro regions. In every case where the function of the pro region has been investigated, it has been found to be necessary for folding and secretion (1). One of the most striking and best studied examples of pro-mediated folding is the bacterial enzyme, ␣-lytic protease (␣LP). ␣LP (EC 3.4.21.12) is a 198-aa serine protease secreted by the Gram-negative soil bacterium Lysobacter enzymogenes to degrade other soil microorganisms. The overall threedimensional fold of ␣LP clearly places it in the same family as the mammalian digestive serine proteases chymotrypsin, trypsin, and elastase, despite only moderate sequence homology (2). In contrast to these mammalian homologues, whose small N-terminal zymogen peptides simply prevent premature activation, ␣LP is synthesized with a large 166-aa N-terminal pro region (Pro) that is required for proper folding of its mature protease domain (3). In vivo, coexpression of ␣LP and Pro, either in cis as the natural precursor molecule or in trans as two separate polypeptide chains results in the secretion of active ␣LP (4), whereas expression of ␣LP alone leads to accumulation of the protease in the outer membrane because of apparent misfolding.In vitro energetic studies reveal a novel means of stability for the mature protease arising from kinetics. This not only distinguishes ␣LP from its mammalian homologues but provides compelling support for the possibility of metastable native conformations in general. Emerging structural and energetic details o...