Study of structure/function relationships constitutes an important field of 
research, especially for modification of protein function and drug design. 
However, the fact that rational design (i.e. the modification of amino acid 
sequences by means of directed mutagenesis, based on knowledge of the 
three-dimensional structure) appears to be much less efficient than irrational 
design (i.e. random mutagenesis followed by in vitro selection) clearly 
indicates that we understand little about the relationships between primary 
sequence, three-dimensional structure and function. The use of evolutionary 
approaches and concepts will bring insights to this difficult question. The 
increasing availability of multigene family sequences that has resulted from 
genome projects has inspired the creation of novel in silico evolutionary 
methods to predict details of protein function in duplicated (paralogous) 
proteins. The underlying principle of all such approaches is to compare the 
evolutionary properties of homologous sequence positions in paralogs. It has 
been proposed that the positions that show switches in substitution rate over 
time--i.e., 'heterotachous sites'--are good indicators of functional divergence. 
However, it appears that heterotachy is a much more general process, since most 
variable sites of homologous proteins with no evidence of functional shift are 
heterotachous. Similarly, it appears that switches in substitution rate are as 
frequent when paralogous sequences are compared as when orthologous sequences 
are compared. Heterotachy, instead of being indicative of functional shift, may 
more generally reflect a less specific process related to the many intra- and 
inter-molecular interactions compatible with a range of more or less equally 
viable protein conformations. These interactions will lead to different 
constraints on the nature of the primary sequences, consistently with theories 
suggesting the non-independence of substitutions in proteins. However, a 
specific type of amino acid variation might constitute a good indicator of 
functional divergence: substitutions occurring at positions that are generally 
slowly evolving. Such substitutions at constrained sites are indeed much more 
frequent soon after gene duplication. The identification and analysis of these 
sites by complementing structural information with evolutionary data may 
represent a promising direction to future studies dealing with the functional 
characterization of an ever increasing number of multi-gene families identified 
by complete genome analysis.