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

alpha-Lytic protease (alpha LP), an extracellular bacterial protease, is synthesized with a large amino-terminal pro-region that is essential for its folding in vivo and in vitro(1,2). In the absence of the proregion, 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 I-1,I-3. Although a basic tenet of protein folding is that the native state of a protein is at the minimum free energy(4), we show here that both the I and fully unfolded states of alpha LP we lower in free energy than the native state. Native alpha LP is thus metastable: its apparent stability derives from a large barrier to unfolding. Consequently the evolution of alpha LP 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.