Volume and polarity changes accompanied by amino acid substitutions in protein evolution

We evaluated the volume and polarity changes accompanied by amino acid substitutions along branches of the phylogenetic trees of cytochrome c, myoglobin and hemoglobin alpha and beta chains. In most cases the volume changes accompanied by the substitutions were found to be much larger than the volume of cavities existing in the interior of X-ray-analysed proteins. This implies that the interior of the proteins is very flexible and the necessary space for a larger amino acid residue substitution can be provided by adjusting nearby structures. Also, the volume and polarity changes are not particularly dependent on whether the substituted site is located in the exterior or interior of the proteins. This result supports the concept of the covarions by Fitch and Markowitz, when combined with the known fact that the exterior sites are more variable than the interior ones during protein evolution.