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.
Highlights
With the exception of unstructured protein domains, the integrity of a protein’s structure and function is largely dependent on its thermodynamic stability
Stability-function tradeoffs became originally evident in enzymes, in the structural tension created by the arrangement of catalytic residues in active sites
Evolution of a new protein function will be driven by mutation of amino acids in key positions
Summary
With the exception of unstructured protein domains, the integrity of a protein’s structure and function is largely dependent on its thermodynamic stability. Evolutionary processes, be they neutral, or adaptive, involve the acquisition of mutations that may affect protein function and/or stability. The relationship between mutational effects, function and stability is crucial to our understanding of the evolutionary dynamics of proteins [1,2,3,4,5,6], and in engineering, designing, and evolving, novel enzymes in the laboratory [7,8,9,10,11,12]. Stability-function tradeoffs became originally evident in enzymes, in the structural tension created by the arrangement of catalytic residues in active sites. The substitution of an enzyme’s key catalytic side chains (typically into alanine) can dramatically increase stability whilst obviously sacrificing activity [16,17,18,19,20,21,22,23]
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