The of can be studied at the levels of nucleotide sequences, primary structures, secondary structures, and . A nucleotide substitution within a gene coding for a protein can be classified as either synonymous substitution or nonsynonymous substitution (1). A synonymous substitution is the so-called silent substitution that never causes amino acid changes. On the other hand, nonsynonymous substitutions alter the amino acid sequence. The ratio (f) of the number of nonsynonymous substitutions (KA) over that of synonymous substitutions ( Ks) can be used as a measure of the functional constraints on a protein. It has constantly been found that the rate of amino acid substitution in the functionally more important parts of the protein is slower than in the less important parts. With the assumption that Ks is a more direct reflection of the mutation rate, because there are no changes of amino acids (although this assumption is not always valid), f is an indicator of the degree of functional constraints for the whole, as well as parts of a protein.
For amino acid sequences, the rate of amino acid substitution is estimated as a measure of an evolutionary rate of change of proteins (for more details, see ). Regarding secondary structures, most functional protein domains are composed of a certain combination of alpha-helix, beta-sheet structures, , and irregular parts. One of the strong driving forces of protein evolution should be , in which those secondary structures may play an important role. and have elucidated a large number of three-dimensional . Moreover, methods for the prediction of tertiary structures of proteins from the amino acid sequences have improved tremendously (2). This provides a unique opportunity for us to make throughout comparisons of tertiary structures among different proteins, possibly leading to the elucidation of the ancient evolution of proteins and genes.
