Fig. 4.

Mg²⁺ induces the partial insertion of loop 3 on both C2 domains. a, b Fluorescence analysis using environmentally-sensitive probe, IAEDANS introduced at the tip of the aliphatic loops of the C2 domains shows the Mg²⁺ (red curve) induces an insertion of loop 3 (residue S235 of C2A & residue I367 of C2B) as evidenced by an increase in the fluorescence intensity and blue-shift in the emission maxima as compared to an EDTA control (black curve). This Mg²⁺-induced membrane interaction is distinct, with shallower positioning of the aliphatic loops in the lipid membrane as compared to the Ca²⁺-bound state (blue curve) (c, d) Normalized fluorescence signal at the emission maxima shows that Ca²⁺-induces deep insertion of both loop 1 (residue M173 in C2A and V304 in C2B) and loop 3, but Mg²⁺ specifically triggers partial insertion of loop 3 alone in both C2 domains. Representative fluorescence curves are shown, along with averages and standard deviations from 4 to 5 independent experiments