Figure 6.

Intermolecular FRET does not reveal syntaxin-dependent structural changes. (A) Structure of full-length SNAP25 constructs containing single fluorophores. These constructs were also tagged with separate epitopes (HA and myc, respectively) for ease of identification of the individual species on blots. (B) SNAP25 constructs shown in A were expressed in HEK293T cells alone or together with or without syntaxin or syntaxin plus VAMP. Membranes were prepared as in Materials and Methods, and spectra were obtained using a spectrofluorimeter. The groups are listed in order of decreasing fluorescence at 526 nm (the order they appear in the figure). The spectrum of myc-sYs-SNAP25 (which contains no cerulean and thus has little emission when excited at 435 nm) was omitted from the figure. This spectrum was used to subtract the component of citrine emission due to excitation at the cerulean wavelength (435 nm) or spill-over, from each group that included myc-sYs-SNAP25. In addition, the expression of the two SNAP25 constructs was examined by SDS-PAGE and protein blotting and was determined to be ∼1:1 in all groups in which they were coexpressed.