On the choice of reference mutant states in the application of the double-mutant cycle method

Pairwise interactions in proteins can be detected and, in certaincircumstances, their strength measured by applying the methodof double-mutant cycles. Such cycles comprise wild type protein,two single mutants and the corresponding double mutant The analysisof double-mutant cycles is most straightforward when the mutationsare to alanine since interactions are mostly removed withoutnew interactions being formed. Here, ‘not-to-alanine’double-mutant cycles are analysed. It is shown that a ‘not-to-alanine’double-mutant cycle can be decomposed into four double-mutantcycles with mutations only to alanine. The coupling energy correspondingto the ‘not-to-alanine’ double-mutant cycle is expressedas a function of the coupling energies of these four cycles.