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spective analyses of natural evolution have uncovered clear examples of mutations whose beneficial effects are contingent on the existence of other initially neutral or even slightly deleterious mutations. The surprising empirical lesson is that such epistasis frequently occurs through a simple mechanism, allowing it to be both easily understood and leveraged for engineering purposes.

Early experimental clues to the common origin of much of protein mutational epistasis came from genetic studies that found that the same “global suppressor” mutations could often remedy the effects of several different deleterious mutations (Shortle and Lin, 1985; Rennell and Poteete, 1989). In many cases, the global suppressor mutations stabilized the protein’s folded structure, suggesting that they compensated for destabilization caused by the deleterious mutations (Shortle and Lin, 1985). The role of stability compensation in adaptive evolution was demonstrated in a study showing that a naturally occurring antibiotic-resistance enzyme acquired activity on new antibiotics by pairing a stabilizing mutation with one or more catalytically beneficial but destabilizing mutations (Wang et al., 2002).

The contribution of directed evolution experiments has been to demonstrate the ubiquity of such stability-mediated epistasis. Introducing just 1 stabilizing mutation into a lactamase enzyme reduced the fraction of random single amino acid mutations that inactivated the protein by one-third (Bloom et al., 2005). A cytochrome P450 enzyme that had been engineered to contain a handful of stabilizing mutations was nearly twice as tolerant to random mutations (Bloom et al., 2006). And a thermostable chorismate mutase was a remarkable 10-fold more tolerant to randomization of a large helical region than its mesostable counterpart (Besenmatter et al., 2007). The extensive stability-mediated epistasis suggested by these experiments can be visualized in terms of a protein stability threshold, as illustrated in Fig. 8.4. In a directed evolution experiment, stability is under selection only insofar as the protein must fold to its proper 3D structure with sufficient stability to perform the target biochemical function. Mutations that increase or decrease stability are therefore neutral as long as the protein remains more stable than some threshold value. But because most mutations are destabilizing, an initially neutral stabilizing mutation can increase a protein’s robustness to other, subsequent mutations.

Directed evolution has shown the crucial role that stability-based epistasis can play in adaptive evolution. One experiment directly compared the frequency with which a marginally stable and a highly stable cytochrome P450 enzyme could acquire activities on a set of new substrates upon random mutation. Libraries of mutants of both enzymes were screened, and a markedly higher fraction of mutants of the stable protein were found to exhibit the new activities (Bloom et al., 2006). This increased evolvability of the stable enzyme was caused by its ability to better toler-

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