Fig. 5: Sec18/NSF does not unfold the Habc domain.
a, Diagram of the smFRET disassembly and reassembly assay with NSF/α-SNAP and linked neuronal SNAREs. Either the linked SNARE complex is stochastically labeled (residue number 249 in syntaxin and residue number 82 in synaptobrevin) or the syntaxin Habc domain is stochastically labeled (residue numbers 35 and 105 in the Habc domain). b, Representative time trace of labeled linked SNARE complex in the nonhydrolyzing condition. Red represents the acceptor and green represents the donor dye fluorescence intensity time traces. The black line represents fitting by a vbGMM (n = 53). Blue represents the FRET efficiency. The right sub panels are the respective probability distributions. c, Representative time trace of labeled SNARE complex in disassembly conditions. Red represents the acceptor and green represents the donor dye fluorescence intensity time traces. The black line represents fitting by a vbGMM51 (n = 114). Blue represents the FRET efficiency. The right sub panels are the respective probability distributions. d, Representative time trace of labeled Habc domain in the nonhydrolyzing condition. Green represents the donor and red represents the acceptor dye fluorescence intensity time traces. The black line represents fitting by a vbGMM (n = 27). Blue represents the FRET efficiency. The right sub panels are the respective probability distributions. e, Representative time trace of labeled Habc domain in disassembly conditions. Green represents the donor and red represents the acceptor dye fluorescence intensity time traces. The black line represents fitting a vbGMM (n = 22). Blue represents the FRET efficiency. The right sub panels are the respective probability distributions. f, Histograms of E-FRET for all four conditions. The blue dotted line represents a Gaussian fit of the data. The y axis is the normalized frequency of occurrence of the E-FRET states in the traces. Top left, SNARE E-FRET for the nonhydrolyzing condition; top right, SNARE E-FRET for the hydrolyzing condition; bottom left, Habc E-FRET for the nonhydrolyzing condition; bottom right, Habc E-FRET for the hydrolyzing condition. g, Native gel of labeled uncrosslinked or crosslinked yeast SNARE complex labeled with Oregon green maleimide 488. h, Quantification of gel densitometry of n = 3 independent disassembly experiments. Error bars represent the s.d. of the mean. Densitometries were all normalized to 0-min values.
