Fig. 5.

The Syt1 C₂ domains have similar membrane affinities and cooperate in membrane binding. (A) Titrations of 100 nM Syt1 C₂A domain, C₂B domain and C₂AB* fragment (the * in C₂AB* denotes that the fluorescent probe is attached to the C₂B domain) with liposomes lacking PIP₂ in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. (B) The same titrations shown in A but changing the x axis to better show the points of the titrations obtained at low liposome concentrations. (C) Liposome titrations of 100 nM WT Syt1 C₂AB* fragment and mutant fragments where the Ca²⁺-binding sites of the C₂A domain (C₂aB*) or C₂B domain (C₂A*b) were disrupted with D178A,D230A,D232A or D309A,D363A,D365A mutations. All of the data were fit to a Hill function.