Mutant cycle analysis for a multistate protein. The WT protein with unperturbed sites (XY) activates via multiple intermediate states, which enables the protein to switch between the initial conformation, S₁, and the final conformation S_n+1, which is driven by an external physical or chemical driving force (stimulus). The two single mutants (X0) or (0Y) and the double mutant (00) follow a similar activation scheme. The mutations can affect one or multiple transitions of the scheme, which might not be known a priori. In this case, the free energy of perturbation is calculated from the measurements of conjugate displacement associated with the transition from S₁ to S_n+1.