Decoupled folding and functional landscapes allow alpha-lytic protease and Streptomyces griseus protease B to develop novel native-state properties

Most secreted bacterial proteases, including Streptomyces griseus protease B (SGPB) and alpha-lytic protease (alphaLP), are synthesized with covalently attached pro regions necessary for their folding. The previously characterized alphaLP folding landscape suggested that this requirement is a result of its remarkable folding mechanism, where a very large free energy barrier for folding is a consequence of a functionally important high unfolding free energy barrier. I characterized the SGPB folding landscape to better understand the determinants for the folding of this striking family of proteins. We find that SGPB, like alphaLP, derives its stability kinetically, from an extremely slow and highly cooperative unfolding transition. These unique unfolding transitions extend their functional lifetimes beyond that seen for trypsin, a thermodynamically stable protease, by increasing their resistance to exogenous proteolysis. The price for evolving kinetic stability is remarkably large: each factor of 2.4--8 in protease resistance is accompanied by a cost of ∼105 in the spontaneous folding rate and ∼5--9 kcal/mol in thermodynamic stability. These penalties have necessitated the co-evolution of increasingly effective pro region folding catalysts. Nevertheless, it remains unknown how these distinctive unfolding transitions are achieved. I performed a quasi-thermodynamic analysis of the SGPB unfolding transition and compared it to the analogous data for alphaLP This revealed that SGPB and alphaLP unfold through similar mechanisms, characterized by large change in heat capacity and large unfolding free energies of activation that occur at physiological temperatures. SGPB and alphaLP are distinguished from thermodynamically stable proteins by their simultaneous optimization of all three parameters. This is again associated with large costs for folding: alphaLP violates a universal behavior observed for all thermodynamically stable proteins studied. Additionally, I investigated the structural origins of the unique folding of these proteases through mutagenesis. To determine the role of a conserved beta-hairpin in the alphaLP folding mechanism, I replaced the alphaLP beta-turn with the more favorable SGPB beta-turn. Characterization of the folding of this variant revealed that packing of the beta-hairpin is important for developing LP's kinetic stability. Additionally, it highlights the restrictions imposed by the concurrent evolution of folding and functional properties.