Person:

Dasmeh, Pouria

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 (ωML) 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.

Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10–20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the (O_2)-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8–1.2 (µM^{−1})), folding stabilities of cetacean Mbs are ∼2–4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and Bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds.