The Influence of Selection for Protein Stability on dN/dS Estimations

Understanding the relative contributions of various evolutionary processes—purifying selection, neutral drift, and adaptation—is fundamental to evolutionary biology. A common metric to distinguish these processes is the ratio of nonsynonymous to synonymous substitutions (i.e., dN/dS) interpreted from the neutral theory as a null model. However, from biophysical considerations, mutations have non-negligible effects on the biophysical properties of proteins such as folding stability. In this work, we investigated how stability affects the rate of protein evolution in phylogenetic trees by using simulations that combine explicit protein sequences with associated stability changes. We first simulated myoglobin evolution in phylogenetic trees with a biophysically realistic approach that accounts for 3D structural information and estimates of changes in stability upon mutation. We then compared evolutionary rates inferred directly from simulation to those estimated using maximum-likelihood (ML) methods. We found that the dN/dS estimated by ML methods (ω(ML)) is highly predictive of the per gene dN/dS inferred from the simulated phylogenetic trees. This agreement is strong in the regime of high stability where protein evolution is neutral. At low folding stabilities and under mutation-selection balance, we observe deviations from neutrality (per gene dN/dS > 1 and dN/dS < 1). We showed that although per gene dN/dS is robust to these deviations, ML tests for positive selection detect statistically significant per site dN/dS > 1. Altogether, we show how protein biophysics affects the dN/dS estimations and its subsequent interpretation. These results are important for improving the current approaches for detecting positive selection.