Proteins are functional units of the living world and they share great diversity in structure, function and evolutionary history. Molecular weight is one of the most basic physicochemical properties of proteins with implications in protein stability, interactions and subcellular localization. The relationship between molecular-weight variation and phylogenetic divergence, however, in each of the functionally distinct protein families is still poorly understood. This study compares 20 commonly studied proteins which are representatives of 6 functional classes of proteins including: structural (Actin, Myosin, Collagen, Keratin), enzymatic (Catalase, Amylase, Lipase, Pepsin, Trypsin, Urease, Lysozyme, Ribonuclease), transport (Hemoglobin, Albumin, Transferrin), regulatory (Insulin), immune (Immunoglobulin), clotting (Fibrinogen), storage (Casein), and electron transfer (Cytochrome c) proteins across representative vertebrate species.The protein sequences were obtained from the National Center for Biotechnology Information (NCBI) database, and the molecular weights were computed using standard bioinformatics tools. Multiple sequence alignment to find conserved motifs, variable domains and lineage specific insertions or deletions. Maximum likelihood and neighbor-joining phylogenetic trees were used to determine evolutionary relationships between the selected proteins. The topologies obtained were then compared with the molecular weight data to determine if proteins with similar function have convergent mass profiles or if phylogenetic distance outweighs functional constraints in the determination of molecular weight diversity.Initial studies indicate that structural proteins have relatively invariant molecular masses, with very little inter-species variation, indicating relatively strong constraints on maintaining mass for mechanical integrity. The molecular weight diversity of enzymatic and regulatory proteins is, by contrast, significant due to domain shuffling, alternative splicing and species specific adaptations of catalytic efficiency. Transport proteins fall in the middle with fluctuations in their molecular weight matching the needs for their ligands and circulation in vertebrates. Interestingly, some proteins like Cytochrome c and Ribonuclease are very well conserved in molecular weight, adding to their status as molecular chronometers. In contrast, adaptive immune diversification is revealed by the high weights of proteins such as Immunoglobulin, while hemostasis is specialized in the proteins such as Fibrinogen.The molecular weight of a species does not always reflect its species phylogeny, as molecular constraints often have greater influence than evolutionary distance, especially in enzymes with a strictly defined size requirement in their active site. For proteins of less functional rigidity, however, the molecular weight divergence increases with phylogenetic branching, so that there is a positive correlation between the two quantities, which is consistent with a neutral drift model interrupted by episodic selection. The value of this research lies in the way it demonstrates how useful it is to combine the two approaches of phylogenetics and biophysical parameters to gain insight into the evolutionary paths of proteins. The results help gain a better understanding of the link between sequence evolution and molecular traits of proteins in their phenotypes, and also offer the possibility to predict protein behavior between species. Also, the data produced here provide a useful resource for future studies in biomolecular engineering, protein structure-function relationships, and protein evolution studies. Finally, using a phylogenetic framework, it has been conclusively demonstrated that molecular weight is a useful, but complex, tool for measuring evolutionary constraint, functional specialization, and adaptive divergence for common proteins in vertebrates.
