Rearrangements under confinement lead to increased binding energy of Synaptotagmin‐1 with anionic membranes in Mg2+ and Ca2+.

Synaptotagmin‐1 (Syt1) is the primary calcium sensor (Ca²⁺) that mediates neurotransmitter release at the synapse. The tandem C2 domains (C2A and C2B) of Syt1 exhibit functionally critical, Ca²⁺‐dependent interactions with the plasma membrane. With the surface forces apparatus, we directly measure the binding energy of membrane‐anchored Syt1 to an anionic membrane and find that Syt1 binds with ~6 <italic>k</italic>_B<italic>T</italic> in EGTA, ~10 <italic>k</italic>_B<italic>T</italic> in Mg²⁺ and ~18 <italic>k</italic>_B<italic>T</italic> in Ca²⁺. Molecular rearrangements measured during confinement are more prevalent in Ca²⁺ and Mg²⁺ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. These results provide energetic and mechanistic details of the Syt1 Ca²⁺‐activation process in synaptic transmission. [ABSTRACT FROM AUTHOR]