Fig. 3: Vamp anchored SNARE-nanodisc disassembly by NSF monitored using three-colour single-molecule FRET. | Nature Communications

Fig. 3: Vamp anchored SNARE-nanodisc disassembly by NSF monitored using three-colour single-molecule FRET.

From: Single-molecule two- and three-colour FRET studies reveal a transition state in SNARE disassembly by NSF

Fig. 3: Vamp anchored SNARE-nanodisc disassembly by NSF monitored using three-colour single-molecule FRET.

a Procedure of the smFRET assay of SNARE disassembly by NSF using TIRF microscope. b Pictorial representation of complexes on the glass slide before and after the disassembly by NSF. c Relative percentage of spots detected in the Cy3 channel under various experimental conditions. SNARE, NVamp-anchored SNARE containing NStx-Cy3, NSN25-Cy5 and NVamp-Cy7 immobilised on to the TIRFM surface; NSF activity, After the addition of α-SNAP, NSF along with 2 mM ATP and 2 mM MgCl2; No Mg2+, After the addition of α-SNAP, NSF along with 2 mM ATP, without MgCl2; No extra ATP, After the addition of α-SNAP, NSF complexed with ATP along with 2 mM MgCl2; No NSF, After the addition of α-SNAP, 2 mM ATP and 2 mM MgCl2; No α-SNAP, After the addition of NSF, 2 mM ATP and 2 mM MgCl2. The data shows mean ± SD from multiple experiments from 4 independent sample preparations with multiple (n = 3-6) SNARE disassembly experiments. p = ***< 0.005 and **< 0.01, two-sided paired t test. d Percentage of the classified traces based on the chain disassembly time. n = 4, each n is data from an individual sample preparation, with 2–4 experiments from each sample. Values are mean ± SD. e, f Single-molecule trajectories of the NStx-Cy3 (green), NSN25-Cy5 (red) and NVamp-Cy7 (dark grey) during the disassembly. e a representative trace showing both NStx-Cy3 and NSN25-Cy5 chains disassembling from NVAMP-Cy7 at the same time. f a representative trace where NStx-Cy3 separation is observed before it disassembles first, followed by NSN25-Cy5 dissociation. g Cumulative distribution of the lifetime of the transient NStx-Cy3 separated state (n = 84 traces). h Pictorial representation of two major pathways of SNARE disassembly by NSF under our experimental design. Pathway I, showing both NStx and NSN25 disassembly together from NVamp or vice versa (40%) and Pathway II, with an NStx separated transient state observed during NStx disassembly first, followed by NSN25 (50.5%).

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