Selection enhances protein evolvability by increasing mutational robustness and foldability

Selection enhances mutation toleration Mutations generate variability that is either neutral or subject to natural selection. Robustness is a measure of the ability to withstand deleterious mutational effects. Zheng et al. exposed Escherichia coli populations expressing a yellow fluorescent protein to strong, weak, or no selection for yellow fluorescence for four generations. They then selected these populations to a related function, green fluorescence, for four more generations. The strong selection first for yellow and then green fluorescence resulted in the most green fluorescence and the accumulation of the most mutations. This outcome likely was due to the increased foldability of the protein. Selection thus provides a threshold for mutation accumulation, but robustness maintains a buffer necessary for protein evolution. Science, this issue p. eabb5962 Directed evolution of yellow fluorescent protein into green fluorescent protein shows how selection can improve functional evolvability. INTRODUCTION Natural selection plays a central role in adaptive evolution, but we still know little about its role in changing evolvability—the ability to bring forth new and adaptive phenotypes. Different kinds of selection may increase evolvability by different means. Weak purifying selection may enhance evolvability by promoting the accumulation of neutral or slightly deleterious mutations that can serve as stepping stones toward new phenotypes. By contrast, strong directional selection may enhance evolvability by favoring the accumulation of beneficial mutations that can enhance both fitness and evolvability, such as mutations that increase a protein’s thermodynamic stability or its robustness to mutations. RATIONALE To find out how the strength of selection affects protein evolvability, we subjected populations of yellow fluorescent proteins to multiple rounds of directed evolution in Escherichia coli. To control the strength of selection with precision, we used high-throughput phenotypic screening via fluorescence-activated cell sorting. During a first phase of our experiment (phase I), we subjected our populations to either strong selection, weak selection, or no selection on the ancestral phenotype of yellow fluorescence. During the second phase (phase II), we evolved all populations under the same selection pressure toward the new phenotype of green fluorescence. We subsequently used high-throughput phenotypic screening to study how phenotypes evolved in all our populations. In every generation, we also studied genotypic evolution with single-molecule real-time sequencing. We then engineered key adaptive mutants and determined their phenotype and thermodynamic stability. In addition, we determined the robustness of their phenotype to DNA mutations. Furthermore, we quantified the foldability of these mutants by unfolding them and observing their refolding kinetics. RESULTS We found that populations under strong selection for the ancestral yellow fluorescent phenotype during phase I subsequently evolved the new green fluorescent phenotype most rapidly during phase II. Compared to populations under weak or no selection, they reached higher green fluorescence during each generation of phase II and evolved a green emission peak more rapidly. Strong selection promoted both the elimination of deleterious mutations and the accumulation of foldability-improving mutations. As a result, proteins under strong selection evolved higher efficiency of protein folding (foldability) and, to an even greater extent, higher robustness to mutations than proteins under weak or no selection. Their robustness and foldability accelerated the selective sweeps of neofunctionalizing mutations that are necessary to evolve a new phenotype. By contrast, proteins under weak selection accrued more deleterious mutations that slowed down the fixation of neofunctionalizing mutations during the evolution of the new phenotype, even though neofunctionalizing mutations had initially risen to higher frequencies under weak selection. CONCLUSION Strong directional selection enhances the evolvability of a new phenotype to a greater extent than weak purifying selection. The responsible mutations enhance tolerance to mutations, improve protein foldability, and thus increase accessibility of a protein’s native state. In doing so, they promote the formation of correctly folded states that can display new functions after incorporating neofunctionalizing mutations. Although “first order” selection of fitness-enhancing mutations can be in conflict with “second-order” selection of evolvability-enhancing mutations, our experiments demonstrate a class of mutations that avoid this conflict, because the mutations they reveal enhance both fitness and evolvability. In the context of an adaptive landscape (see figure), they do so by circumnavigating rather than traversing adaptive valleys, passing through flat regions of such a landscape, and thus allowing an evolving population to climb a new adaptive peak more rapidly. More generally, our experiments prove that natural selection itself can create the conditions under which Darwinian evolution can succeed. Selection can drive evolvability. Evolutionary theory holds that Darwinian evolution takes place on adaptive landscapes of fitness, which can be visualized as topological maps of high-fitness peaks and low-fitness valleys. This hypothetical landscape illustrates how mutations can increase evolvability by enhancing both fitness and mutational robustness. Favored by strong selection because they enhance fitness, such mutations move an evolving population into a region of low curvature and high robustness (red arrow), from which the population can bypass rather than traverse (blue arrow) an adaptive valley on its way to an adaptive peak. Natural selection can promote or hinder a population’s evolvability—the ability to evolve new and adaptive phenotypes—but the underlying mechanisms are poorly understood. To examine how the strength of selection affects evolvability, we subjected populations of yellow fluorescent protein to directed evolution under different selection regimes and then evolved them toward the new phenotype of green fluorescence. Populations under strong selection for the yellow phenotype evolved the green phenotype most rapidly. They did so by accumulating mutations that increase both robustness to mutations and foldability. Under weak selection, neofunctionalizing mutations rose to higher frequency at first, but more frequent deleterious mutations undermined their eventual success. Our experiments show how selection can enhance evolvability by enhancing robustness and create the conditions necessary for evolutionary success.