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. 2017 May 8;36(12):1788–1802. doi: 10.15252/embj.201696270

Figure 5. The syntaxin1:SNAP25:Munc18‐1 is resistant to disassembly by NSF‐αSNAP but becomes sensitive after binding of synaptobrevin.

Figure 5

  1. Double‐labeled syntaxin1:SNAP25:Munc18‐1 complex containing syntaxin1 (labeled with Oregon Green) and SNAP25 (labeled with Texas Red) was purified. Incubation with NSF‐αSNAP in the presence of Mg2+‐ATP did not result in any change of donor fluorescence. Incubation with 1 M NaCl resulted in a small increase in donor fluorescence, indicating that under high‐salt conditions, some minor dissociation of the complex does occur.
  2. Purified, double‐labeled Syx:SN25 (2:1) complex containing syntaxin1 and SNAP25 (labeled as in A) could be disassembled by NSF‐αSNAP as evident from a rapid increase in donor fluorescence (loss of FRET).
  3. Fluorescence anisotropy measurements showing the disassembly of the SNARE complex generated upon addition of fluorescently labeled synaptobrevin to a pre‐assembled syntaxin1:SNAP25:Munc18‐1 complex. Addition of NSF‐αSNAP led to a rapid decrease in the anisotropy as expected for SNARE disassembly. The red arrows indicate the addition of the protein(s)/reagents, whereas the black arrows indicate the reaction condition.