How Protein Stability and New Functions Trade Off

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.

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

“…This was unexpected, given that KE59 design was based on a scaffold of hyperthermophilic enzyme, and the design process involved protein free energy minimization. Because mutations are destabilizing on average and function-modifying mutations tend to be more destabilizing than neutral ones (18,19), proteins with marginal stability show low tolerance to mutations and hence limited evolvability (20,21). Indeed, our attempts of directed evolution of unstable KE designs such as KE59 were unsuccessful.…”

“…The stabilizing mutations were located at the protein surface, either in the loops or helices, and they had little effect on the kinetic parameters of KE59 (Table 1). Following another two rounds of directed evolution, there was a decrease in the expression of the evolved variants due to the destabilizing effect of function-modifying mutations (19). Therefore, at Round 5, consensus mutations were included again and combinations that were not observed in the first stabilization attempt were incorporated.…”

Stability-activity tradeoffs constrain the adaptive evolution of RubisCO

“…3). This small amplitude of the effects of mutation observed in nature suggests that RubisCO evolves within a small island of stability (3,5).…”

“…Compensatory mutations are then needed to restore global stability. These processes are referred to as stability-activity tradeoffs (5)(6)(7). Furthermore, proteins with higher stability confer greater evolvability, because there is more scope to accept destabilizing yet functionally beneficial changes (8).…”

The Effects of Stabilizing and Directional Selection on Phenotypic and Genotypic Variation in a Population of RNA Enzymes

“…For example, its activity-based phenotype (fraction of ribozyme reacted) can be affected by several fundamental properties of RNA structure and catalysis, including RNA folding, thermodynamic stability of the catalytically active structure, forward and reverse rates of catalysis, and affinity of the ribozyme for the substrate and product of the reaction. Because usually not all of these properties can be simultaneously optimized in any one molecule, many mutations in enzymes have pleiotropic effects, just as in more complex systems (Wang et al 2002;Tokuriki et al 2008;Soskine and Tawfik 2010). In addition, the structural stability of the Azoarcus ribozyme enables considerable mutational robustness which allows cryptic genetic variation with little phenotypic effect to accumulate in populations (Hayden et al 2011).…”