FIGURE 8.4 The effect of a mutation can depend on the stability of the protein into which it is introduced. As shown here, proteins that are more stable than the threshold can fold and function, whereas those that are less stable than the threshold fail to fold and are therefore nonfunctional. A particular functionally beneficial but destabilizing mutation may therefore only be tolerated by a protein that has previously accumulated one or more stabilizing substitutions.

ate catalytically beneficial but destabilizing mutations (Bloom et al., 2006). These results indicate that stabilizing mutations increase evolvability by the same mechanism that they increase mutational robustness.

The existence of widespread stability-mediated epistasis further explains why trapping on fitness peaks is not an important concern in directed protein evolution, although it does emphasize a role for neutral mutations. A protein that has been pushed to the margins of tolerable stability may lose access to functionally beneficial but destabilizing mutations. But this protein is still not stuck on a fitness peak, because it can regain its mutational robustness and evolvability by accumulating initially neutral but stabilizing mutations. In a nondirected context, such a process might require a time-consuming wait for stabilizing mutations to spread by neutral drift. But in a directed evolution experiment, the process can be expedited by intentional selection for stabilizing mutations, as was done in the cytochrome P450 experiment described above.

Directed protein evolution experiments have demonstrated that once a biochemical function is present at even a low level, it can usually be