Schematic depiction of FMC and GIA using energy profile diagrams. Each energy profile in the thermodynamic mutant cycle represents a three-state sequential gating process involving two closed states (C₀ and C₁) and an open state (O). One of the mutants destabilizes the state C₁ (A₀B₁), and the second mutant stabilizes state C₀ (A₁B₀), but the effect is additive on the double mutant (A₁B₁; i.e., C₁ is destabilized and C₀ is stabilized). The pink arrows show the free-energy difference computed by the G-V curves (as is done in FMC), whereas the blue arrows show the free-energy difference computed by the Q-V curves (as is done in GIA). Note that in GIA, the measured ΔG is that between C₀ and O in all four cases, whereas in FMC, the measured ΔG is between C₁ and O in A₀B₀ but between C₀ and O in the other three.