Approaches to predicting effects of single amino acid substitutions on the function of a protein

The relative activities of 313 mutants of the gene V protein of bacteriophage f1, assayed in vivo, have been used to evaluate two approaches to predicting the effects of single amino acid substitutions on the function of a protein. First, we tested methods that only depend on the properties of the wild-type and substituting amino acids. None of the properties or measures of the functional equivalence of amino acids we tested, including the frequency of exchange of amino acids among homologous proteins as well as changes in side-chain size, hydrophobicity, and charge, were found to be more than weakly correlated with the activities of mutants. The principal reason for this poor correlation was found to be that the effect of a particular substitution varies considerably from site to site. We then tested an approach using the activities of several mutants with substitutions at a site to predict the activity of another mutant, and we find that this is a relatively good indicator of whether the other mutant at that site will be functional. A predictive scheme was developed that combines the weak information from the models depending on the properties of the wild-type and substituting amino acids with the stronger information from the tolerance of a site to substitution. Although this scheme requires no knowledge of the structure of a mutant protein, it is useful in predicting the activities of mutants.