Symmetry facilitated the evolution of heterospecificity and high-order stoichiometry in vertebrate hemoglobin.

Many proteins form paralogous multimers-molecular complexes in which evolutionarily related proteins are arranged into specific quaternary structures. Little is known about the mechanisms by which they acquired their stoichiometry (the number of total subunits in the complex) and heterospecificity (the preference of subunits for their paralogs rather than other copies of the same protein). Here, we use ancestral protein reconstruction and biochemical experiments to study historical increases in stoichiometry and specificity during the evolution of vertebrate hemoglobin (Hb), an αβ heterotetramer that evolved from a homodimeric ancestor after a gene duplication.