Unfolded conformations of α-lytic protease are more stable than its native state

α-Lytic protease (αLP), an extracellular bacterial protease, is synthesized with a large amino-terminal pro-region that is essential for its folding in vivo and in vitro1,2. 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 (>26 kcal mol−1) which separates the native state ‘N’ from I1,3. Although a basic tenet of protein folding is that the native state of a protein is at the minimum free energy4, we show here that both the I and fully unfolded states of αLP are lower in free energy than the native state. Native αLP is thus metastable: its apparent stability derives from a large barrier to unfolding. Consequently, the evolution of αLP 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.