Figure 1.

Calcium-dependent calmodulin binding to GST-VAMP 2_1–96. (A) Bacterially expressed GST-fusion proteins containing SNAP-25, the cytoplasmic domains of syntaxin 1A, VAMP 2, and GST (2 μM) were incubated with calmodulin–agarose (5 μM) in TBS in the presence of 1 mM CaCl₂ or 5 mM EDTA. After extensive washing, bound proteins were eluted with sample buffer and analyzed by SDS/PAGE and Coomassie blue staining. (B) GST-VAMP 2_1–96 was diluted into running buffer containing 1 mM CaCl₂ and injected on a streptavidin-coated sensorchip loaded with biotinylated calmodulin. An increase in resonance occurred during VAMP 2 injection (association), followed by decrease at the end of the injection period (dissociation). Injection of EDTA induced an abrupt return to baseline (regeneration). Experiments with a range of VAMP 2 concentrations allowed calculation of the rate constants and K_d reported in the text.