Gag-binding activity of AnkGAG1D4 analyzed by microcalorimetry (ITC): affinity and specificity. The affinity of AnkGAG1D4 towards its Gag substrate H6MA-CA (leftmost panel) and the affinity of AnkA32D3 towards its αRep-A3 substrate (rightmost panel) were determined by ITC measurements of ankyrin/substrate mixtures at various protein ratios. The Gag-binding specificity was evaluated by ITC analysis of the mixture of AnkGAG1D4 and irrelevant substrate αRep-A3 at various protein ratios (middle panel). The parameters of each binding reaction, Kd and stoichiometry (N), are shown under the corresponding panel. NA, calculation not applicable.