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 (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. 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

Positively selected sites in cetacean myoglobins contribute to protein stability.

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.Chemistry and Chemical Biolog

Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R g ~1.48-1.51 nm, helicity ~75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0-3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2-11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations

Implications of the model relating to experimental observations.

<p>Implications of the model relating to experimental observations.</p

Experimental Data for Myoglobin States from the Literature.

<p>^a For HoloMb, average based on crystal structures 1A6M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-Ozdemir1" target="_blank">[84]</a>, 1MBO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-Hirst2" target="_blank">[85]</a>, 1U7S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-VanDerSpoel1" target="_blank">[65]</a>, and 1U7R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080308#pone.0080308-VanDerSpoel1" target="_blank">[65]</a>.</p><p>^b Estimated from NMR data, see text.</p

Selection spaces <i>s</i>_i (fitness-differences normalized to wild-type fitness) for mutations causing increased mutant protein expression (<i>A</i>_i,mutant).

<p>(<b>A</b>) Selection acts against increased protein abundance of mutant vs. wild-type (<i>A</i>_i,WT) (Default values of parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090504#pone-0090504-t001" target="_blank">Table 1</a>). (<b>B</b>) High-turnover proteins (with large values of <i>k</i>_d,i) are under stronger selection pressure to perform optimally. (<b>C</b>) For a high-turnover protein (life time ∼1 minute, <i>k</i>_d = 0.01 s⁻¹, larger proteins are under stronger selection to perform optimally, <i>ceteris paribus</i>. (<b>D</b>) Selection pressure is stronger for proteins that are synthetically expensive, as measured by <i>C</i>_s,i (<i>k</i>_d = 0.01 s⁻¹).</p