Figure 1.

Doc2β penetrates membranes in response to Ca²⁺. A, schematic diagrams of syt1 and Doc2β. MID, munc13-interacting domain; TMD, transmembrane domain. B, model illustrating the putative membrane penetration activity of Doc2 where the distal tip of Ca²⁺-binding loop 1 was mutated to cysteine and labeled with the fluorescent dye NBD, shown at right. The shaded stripe in the bilayer leaflet depicts the approximate distribution of the quenching nitroxide on 12-doxyl-PC. Ribbon diagrams show C2A (PDB code 4LCV) and C2B (PDB code 4LDC) of Doc2β from Giladi et al. (52). C, NBD emission spectra from each of the four Ca²⁺-binding loops of Doc2β C2AB. Graph titles indicate the C2 domain and loop labeled (e.g. C2A*(1)-C2B corresponds to loop 1 of C2A, and C2A*(3)-C2B corresponds to loop 3 of C2A). Labeled C2AB was combined with liposomes (15% PS, 30% PC, 20% PE, and 35% cholesterol) in 500 μm EGTA after which Ca²⁺ was added (250 μm free [Ca²⁺]). Ca²⁺ triggered an intensity increase and blue shift in the emission spectra at all four labeling sites, suggesting burial of the probe into the bilayer. Membrane insertion was confirmed with the use of liposomes containing 15% 12-doxyl-PC, which efficiently quenched the fluorescence at each labeled site. Spectra are representative of data from at least four independent trials.