Ca²⁺ binding to the isolated wild-type (WT) C₂A domain of synaptotagmin 1 and the D232N and D238N point mutants of the C₂A domain was monitored by¹H–¹⁵N HSQC acquired with 150–165 μm of purified recombinant C₂A domains. Dissociation constants were calculated from the Ca²⁺titrations as described (Fernández-Chacón et al., 2001) and are expressed in micromolar Ca²⁺.

↵F1-a Calculated from the Ca²⁺ dependence of the ¹⁵N chemical shift of the K200 NH resonance between 0 and 600 μm total Ca²⁺.

↵F1-b Calculated from the Ca²⁺ dependence of the ¹H chemical shift of the S235 NH resonance.

↵F1-c Only a lower limit can be estimated because the site was not saturated at the highest Ca²⁺ concentration used (40 mm).

↵F1-d Estimated based on the fact that no cross-peak movement corresponding to this site is observed up to 40 mm Ca²⁺.