Single Reconstituted Neuronal SNARE Complexes Zipper in Three Distinct Stages
Ying Gao,
Sylvain Zorman,
Gregory Gundersen,
Zhiqun Xi,
Lu Ma,
George Sirinakis,
James E. Rothman*, and
Yongli Zhang*
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
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Fig. 1. Dynamic disassembly and reassembly of the single SNARE complex. (A) Experimental setup. The SNARE complex contains the N-terminal (NTD) and C-terminal (CTD) SNARE domains, with the corresponding VAMP2 regions designed as Vn and Vc, respectively, separated by the ionic layer and the linker domain (LD). (B) Force-extension curve (FEC) of the SNARE-DNA conjugate. The FEC corresponds to the first of five cycles of pull (black) and relaxation (gray) shown in fig. S2. Different segments of the FEC can be fit by the worm-like chain model (red dashed lines), revealing the structures of SNARE assembly states (inset, same red numbering throughout the figures). The LD and CTD transitions are marked by dashed and solid ovals, respectively. (C) Time-dependent extension corresponding to the pulling phase from 8.6 to 17.5 pN (fig. S3). (D) Extension transitions of LD (bottom) and CTD (top) with their idealized transitions determined by the HMM analysis (red traces). The histogram distributions of extension are shown in fig. S5. (E) Structural model of the force-dependent half-zippered state with Vc unzipped to the ionic layer (red).
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Fig. 2. SNAP-25-dependent SNARE assembly. (A) Force-dependent unzipping probability of CTD measured on a single SNARE complex. (B) Corresponding CTD transition rates. Theoretical predictions are shown in lines. (C) FECs measured in the absence (–SNAP-25) and presence (+SNAP-25) of SNAP-25 in solution.
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Fig. 3. Disassembly and reassembly of the SNARE complex under N-terminal pulling force. (A) FECs and their segmental fit (red dashed line) showing different assembly states (inset) including the completely unfolded state 4'. The full SNARE reassembly (cyan arrow) is t-SNARE dependent. (B) Extension-time trace corresponding to the region marked in the dashed oval in (A).
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Fig. 4. Sketches of the energy landscapes for SNARE zippering in the absence (black) and presence of the opposing force load from membranes with (light gray) and without (gray) complexin clamp. The contour length of the SNARE complex between the C termini of syntaxin (residue 265) and VAMP2 (residue 92) is chosen as a reaction coordinate. Error bars show the SDs of the measurements.
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