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 certain circumstances, their strength measured by applying the method of double-mutant cycles, Such cycles comprise wild type protein, two single mutants and the corresponding double mutant, The analysis of double-mutant cycles is most straightforward when the mutations are to alanine since interactions are mostly removed without new 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-mutant cycles with mutations only to alanine. The coupling energy corresponding to the 'not-to-alanine' double-mutant cycle is expressed as a function of the coupling energies of these four cycles.