How Protein Stability and New Functions Trade Off

Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (Delta Delta G), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (Delta Delta G>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied Delta Delta G computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to Delta Delta G changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average Delta Delta G = +0.9 kcal/mol), and are almost as destabilizing as the average mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non- adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently silent mutations in regions of the protein that are irrelevant to its function.