Figure 3.

Membrane penetration by full-length syt1 reconstituted into nanodiscs. A, experimental scheme. Full-length syt1 was purified, labeled with NBD on loop 3 of C2A or loop 3 of C2B, and reconstituted into 13-nm-diameter nanodiscs comprising membrane scaffolding protein and POPC (ND-syt1). ND-syt1 was combined with liposomes containing acidic phospholipids in EGTA (500 μm) followed by the addition of Ca²⁺ to assay Ca²⁺-independent and Ca²⁺-dependent membrane penetration activity. B, representative spectra for penetration experiments with ND-syt1. As with syt1 C2AB, Ca²⁺ and acidic phospholipids caused an increase and blue shift in NBD fluorescence. Likewise, in the presence of both PS and PIP₂, Ca²⁺-independent penetration by C2B, but not C2A, was observed. Spectra are representative of results from four independent trials.