Figure 5.

Wild-type syntaxin 1A and s1AYK6 point mutant display similar affinity for the II/III loop of the N-type Ca²⁺ channel in a scintillation proximity displacement assay. ³⁵S-labeled s1A-M267X binding to an immobilized recombinant GST fusion protein incorporating the II/III loop of α_1B. Binding assays were performed in the presence of increasing concentrations of soluble competing proteins: wild-type syntaxin 1A fragment (s1A-M267X, square), a corresponding fragment of mutant s1A-YK6 (triangle), and a control protein (GST, circle). All three proteins were derived from bacterial expression and purified. In all scintillation proximity binding assays, nonspecific binding (determined by binding of ³⁵S-s1A-M267X to immobilized GST) was about 23% of total binding (determined by maximal binding of ³⁵S-s1A-M267X to immobilized GST-II/III loop fragment). The data points show mean ± SEM values for percent binding, expressed as a fraction of maximal binding (n = 3).