Computational analysis of vertebrate myoglobins reveals aggregation resistance in aquatic birds and higher surface hydrophobicity in fish

Myoglobin is the major oxygen carrying protein in vertebrate muscle. Previous studies identified in secondarily aquatic mammalian lineages high myoglobin net charge, which serves to prevent aggregation at the extremely high intracellular myoglobin concentrations found in these species. However, it is unknown how aquatic birds that dive for extended durations prevent myoglobin aggregation at their high intracellular myoglobin concentrations. It is also unknown whether secondarily aquatic lineages reduced the surface hydrophobicity of their myoglobins to prevent aggregation. Here, we used a deep learning-predicted distance-based protein folding algorithm to model the tertiary structures of 302 vertebrate myoglobin orthologs and performed a comparative analysis of their predicted net charge and surface hydrophobicities. The results suggest that aquatic avian divers, such as penguins and diving ducks, evolved highly charged myoglobins to reduce aggregation propensity and allow greater storage of oxygen for extended underwater foraging. High myoglobin net charge was also identified in golden eagles, a species that routinely suffers high-altitude hypoxia. Although no general association was found between myoglobin surface hydrophobicity and intracellular concentration, comparison of predicted net charge and surface hydrophobicities revealed significant differences between major vertebrate classes; bird myoglobins are the most positively charge, reptile myoglobins are the most negatively charged, and the myoglobins of ray-finned fish (Actinopterygii) have higher surface hydrophobicity than those of lobe-finned fish (Sarcopterygii). Our findings indicate the convergent evolution of high myoglobin net charge in aquatic birds and mammals, and offer novel insights into the diversification of myoglobin among vertebrate clades.