Pairwise interaction energies between residues of the interfacial triad using GIA. (A–C) GIA was used to measure the interaction energies between E395-Y485 (A), R394-Y485 (B), and R394-E395 (C). For each pair, the normalized Q-V curves of the single and double (alanine) mutants were measured, from which the V_M was extracted and used to calculate the free energy of perturbation. The thermodynamic cycle for each pair is shown in the inset, where each box corresponds to the WT or single or double mutants, colored according to the curves in each panel. The legends correspond to single-letter codes of the amino acids perturbed. Error bars represent SEM.