Reframing prosegment‐dependent folding and limits on natural protein folding landscapes from an evolutionary perspective

In this perspective, we propose that the folding energy landscapes of model proteases including pepsin and alpha‐lytic protease (αLP), which lack thermodynamic stability and fold on the order of months to millennia, respectively, should be viewed as not evolved and fundamentally distinct from their extended zymogen forms. These proteases have evolved to fold with prosegment domains and robustly self‐assemble as expected. In this manner, general protein folding principles are strengthened. In support of our view, αLP and pepsin exhibit hallmarks of frustration associated with unevolved folding landscapes, such as non‐cooperativity, memory effects, and substantial kinetic trapping. The evolutionary implications of this folding strategy are considered in detail. Direct applications of this folding strategy on enzyme design, finding new drug targets, and constructing tunable folding landscapes are also discussed. Together with certain proteases, growing examples of other folding “exceptions”—including protein fold switching, functional misfolding, and prevalent inability to refold—suggests a paradigm shift in which proteins may evolve to exist in a wide range of energy landscapes and structures traditionally thought to be avoided in nature.