Fig. 5.

Increased fluorescence of NBD probes placed at the bottom of the C₂B domain upon membrane binding. (A and B) Representative fluorescence emission spectra of 0.3-μM samples of NBD-labeled N396C mutant C₂AB (A) or lnC₂AB (B) acquired side by side in the presence of phospholipid vesicles (100-μM lipids) and 1 mM EGTA or 1 mM Ca²⁺ (clustering conditions). (C and D) Analogous spectra acquired under the same conditions but using 1-mM lipids (nonclustering conditions). For each set of experiments in A–D, spectra acquired with identical samples containing unlabeled C₂AB or lnC₂AB were subtracted to remove contributions from light scattering to the observed intensities. The data were then normalized to the maximum fluorescence intensity observed in the absence of Ca²⁺. (E) Quantification of NBD fluorescence increases upon membrane binding. NBD fluorescence emission spectra analogous to those shown in A–D were acquired in triplicate under clustering conditions (C₂AB+ and lnC₂AB+) or nonclustering conditions (C₂AB− and lnC₂AB−). Bars represent averages of the ratios between the NBD fluorescence intensity maxima observed in 1 mM Ca²⁺ and 1 mM EGTA in three separate pairs of experiments performed under identical conditions. Error bars represent SDs.