How Protein Stability and New Functions Trade Off

Top Cited Papers
Open Access
Publisher Website
Google Scholar
Abstract
Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (ΔΔG), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (ΔΔG>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied ΔΔG 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 ΔΔG changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average ΔΔG = +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. To perform its function, a protein must fold into a complex, three-dimensional structure that is maintained by a network of interactions between its amino acid residues. Evolution of a new protein function will be driven by mutation of amino acids in key positions (new-function mutations). Such mutation can also hamper interactions that ensure the stability of a protein's fold—sometimes to a degree that renders the protein non-functional. Indeed, previous studies have noted that the evolution of new enzymatic functions is accompanied by significant losses in protein stability, suggesting a “tradeoff” between acquisition of new enzymatic functions and stability. But since most mutations are destabilizing, we sought to compare new-function mutations with other types of mutations. We performed a comprehensive analysis of the type, location, and stability effects of mutations that have conferred new enzymatic functions in laboratory evolution experiments. We found that stability changes (ΔΔG) of new-function mutations are similar to those of all other mutations, but are weaker than those of mutations that characterize neutral evolutionary changes (mutations that accumulate with no change of structure and function). Our analysis also revealed the important role of neutral (i.e., “non-functional”) mutations in compensating for the destabilizing effects of the “new-function” mutations.