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Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism.

Min D, Kim K, Hyeon C, Cho YH, Shin YK, Yoon TY - Nat Commun (2013)

Bottom Line: When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled.In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force.This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.

View Article: PubMed Central - PubMed

Affiliation: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon 305-701, South Korea.

ABSTRACT
Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for membrane fusion, but its molecular mechanism is still unclear. Here using magnetic tweezers, we observe mechanical responses of a single neuronal SNARE complex under constant pulling force. Single SNARE complexes may be unzipped with 34 pN force. When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 11 pN. Thus, mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.

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Intermediates in the mechanical unzipping of a single SNARE complex.(a–c) Real-time traces showing C-to-N unzipping events of single SNARE complexes. The force was increased from 11 pN to 34 pN with loading rate of 58 pN s−1. The traces show one-step (a,b) or two-step unzipping events (c). (d) Distribution of the first unzipping steps of C-to-N unzipping experiment. Two Gaussian peaks were found at 10.2±0.1 nm (s.e.m.) and 19.4±0.2 nm (s.e.m.) (N=119 events from n=41 SNARE complexes). The s.d. values are 2.25 and 1.83 nm, respectively. The distribution of the time lapse (Δt) was fitted using a single exponential equation with a decay time of 1.7 s (right inset). The anticipated structures of the SNARE complex are also shown. The cylinders represent the α-helical structure and the lines represent unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) The sum of the first and second steps from the two-step unzipping events. The distribution shows one Gaussian peak at 21.2±0.01 nm (s.e.m.) with a s.d. of 1.37 nm (N=12 events from n=7 SNARE complexes). (f,g) Real-time traces showing N-to-C unzipping events of single SNARE complexes. The force was increased from 11  to 34 pN with loading rate of 58 pN s−1. The traces show one-step unzipping with a size of ~10 nm (f) or ~20 nm (g). (h) Distribution of the first unzipping steps of N-to-C unzipping experiment. The distribution shows two Gaussian peaks at 10.0±0.2 nm (s.e.m.) and 21.4±0.2 nm (s.e.m.) (N=84 events from n=25 SNARE complexes). The s.d. values are 2.68 nm and 3.31 nm, respectively. (i) Estimated extensions of a single SNARE complex when it is unzipped up to the specific layers from the respective DNA anchor positions. The extension values expected for C-to-N unzipping experiment are shown in the upper row, and the extensions for N-to-C unzipping experiment are shown in the bottom row. The extension values for the zeroth layer (ionic layer) and the knotted layers are shown in red.
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f2: Intermediates in the mechanical unzipping of a single SNARE complex.(a–c) Real-time traces showing C-to-N unzipping events of single SNARE complexes. The force was increased from 11 pN to 34 pN with loading rate of 58 pN s−1. The traces show one-step (a,b) or two-step unzipping events (c). (d) Distribution of the first unzipping steps of C-to-N unzipping experiment. Two Gaussian peaks were found at 10.2±0.1 nm (s.e.m.) and 19.4±0.2 nm (s.e.m.) (N=119 events from n=41 SNARE complexes). The s.d. values are 2.25 and 1.83 nm, respectively. The distribution of the time lapse (Δt) was fitted using a single exponential equation with a decay time of 1.7 s (right inset). The anticipated structures of the SNARE complex are also shown. The cylinders represent the α-helical structure and the lines represent unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) The sum of the first and second steps from the two-step unzipping events. The distribution shows one Gaussian peak at 21.2±0.01 nm (s.e.m.) with a s.d. of 1.37 nm (N=12 events from n=7 SNARE complexes). (f,g) Real-time traces showing N-to-C unzipping events of single SNARE complexes. The force was increased from 11  to 34 pN with loading rate of 58 pN s−1. The traces show one-step unzipping with a size of ~10 nm (f) or ~20 nm (g). (h) Distribution of the first unzipping steps of N-to-C unzipping experiment. The distribution shows two Gaussian peaks at 10.0±0.2 nm (s.e.m.) and 21.4±0.2 nm (s.e.m.) (N=84 events from n=25 SNARE complexes). The s.d. values are 2.68 nm and 3.31 nm, respectively. (i) Estimated extensions of a single SNARE complex when it is unzipped up to the specific layers from the respective DNA anchor positions. The extension values expected for C-to-N unzipping experiment are shown in the upper row, and the extensions for N-to-C unzipping experiment are shown in the bottom row. The extension values for the zeroth layer (ionic layer) and the knotted layers are shown in red.

Mentions: With the force-jump to 34 pN, we observed three types of unzipping events: one-step unzipping with a step size of ~10 nm (Fig. 2a), one-step unzipping with a larger step size of ~20 nm (Fig. 2b) and sequential two-step unzipping with two-step sizes of ~10 nm each (Fig. 2c) (Supplementary Fig. S5 for more traces and Supplementary Fig. S6 for how we determine the step sizes). When we determined the step sizes of the first unzipping events regardless of whether there were second unzipping events or not, the resultant distribution showed two clear Gaussian distributions with peaks at 10.2 and 19.4 nm (Fig. 2d). In addition, when the first and second steps were summed for the two-step unzipping events, the distribution of this sum showed a single Gaussian distribution with a peak at 21.2 nm (Fig. 2e).


Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism.

Min D, Kim K, Hyeon C, Cho YH, Shin YK, Yoon TY - Nat Commun (2013)

Intermediates in the mechanical unzipping of a single SNARE complex.(a–c) Real-time traces showing C-to-N unzipping events of single SNARE complexes. The force was increased from 11 pN to 34 pN with loading rate of 58 pN s−1. The traces show one-step (a,b) or two-step unzipping events (c). (d) Distribution of the first unzipping steps of C-to-N unzipping experiment. Two Gaussian peaks were found at 10.2±0.1 nm (s.e.m.) and 19.4±0.2 nm (s.e.m.) (N=119 events from n=41 SNARE complexes). The s.d. values are 2.25 and 1.83 nm, respectively. The distribution of the time lapse (Δt) was fitted using a single exponential equation with a decay time of 1.7 s (right inset). The anticipated structures of the SNARE complex are also shown. The cylinders represent the α-helical structure and the lines represent unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) The sum of the first and second steps from the two-step unzipping events. The distribution shows one Gaussian peak at 21.2±0.01 nm (s.e.m.) with a s.d. of 1.37 nm (N=12 events from n=7 SNARE complexes). (f,g) Real-time traces showing N-to-C unzipping events of single SNARE complexes. The force was increased from 11  to 34 pN with loading rate of 58 pN s−1. The traces show one-step unzipping with a size of ~10 nm (f) or ~20 nm (g). (h) Distribution of the first unzipping steps of N-to-C unzipping experiment. The distribution shows two Gaussian peaks at 10.0±0.2 nm (s.e.m.) and 21.4±0.2 nm (s.e.m.) (N=84 events from n=25 SNARE complexes). The s.d. values are 2.68 nm and 3.31 nm, respectively. (i) Estimated extensions of a single SNARE complex when it is unzipped up to the specific layers from the respective DNA anchor positions. The extension values expected for C-to-N unzipping experiment are shown in the upper row, and the extensions for N-to-C unzipping experiment are shown in the bottom row. The extension values for the zeroth layer (ionic layer) and the knotted layers are shown in red.
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f2: Intermediates in the mechanical unzipping of a single SNARE complex.(a–c) Real-time traces showing C-to-N unzipping events of single SNARE complexes. The force was increased from 11 pN to 34 pN with loading rate of 58 pN s−1. The traces show one-step (a,b) or two-step unzipping events (c). (d) Distribution of the first unzipping steps of C-to-N unzipping experiment. Two Gaussian peaks were found at 10.2±0.1 nm (s.e.m.) and 19.4±0.2 nm (s.e.m.) (N=119 events from n=41 SNARE complexes). The s.d. values are 2.25 and 1.83 nm, respectively. The distribution of the time lapse (Δt) was fitted using a single exponential equation with a decay time of 1.7 s (right inset). The anticipated structures of the SNARE complex are also shown. The cylinders represent the α-helical structure and the lines represent unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) The sum of the first and second steps from the two-step unzipping events. The distribution shows one Gaussian peak at 21.2±0.01 nm (s.e.m.) with a s.d. of 1.37 nm (N=12 events from n=7 SNARE complexes). (f,g) Real-time traces showing N-to-C unzipping events of single SNARE complexes. The force was increased from 11  to 34 pN with loading rate of 58 pN s−1. The traces show one-step unzipping with a size of ~10 nm (f) or ~20 nm (g). (h) Distribution of the first unzipping steps of N-to-C unzipping experiment. The distribution shows two Gaussian peaks at 10.0±0.2 nm (s.e.m.) and 21.4±0.2 nm (s.e.m.) (N=84 events from n=25 SNARE complexes). The s.d. values are 2.68 nm and 3.31 nm, respectively. (i) Estimated extensions of a single SNARE complex when it is unzipped up to the specific layers from the respective DNA anchor positions. The extension values expected for C-to-N unzipping experiment are shown in the upper row, and the extensions for N-to-C unzipping experiment are shown in the bottom row. The extension values for the zeroth layer (ionic layer) and the knotted layers are shown in red.
Mentions: With the force-jump to 34 pN, we observed three types of unzipping events: one-step unzipping with a step size of ~10 nm (Fig. 2a), one-step unzipping with a larger step size of ~20 nm (Fig. 2b) and sequential two-step unzipping with two-step sizes of ~10 nm each (Fig. 2c) (Supplementary Fig. S5 for more traces and Supplementary Fig. S6 for how we determine the step sizes). When we determined the step sizes of the first unzipping events regardless of whether there were second unzipping events or not, the resultant distribution showed two clear Gaussian distributions with peaks at 10.2 and 19.4 nm (Fig. 2d). In addition, when the first and second steps were summed for the two-step unzipping events, the distribution of this sum showed a single Gaussian distribution with a peak at 21.2 nm (Fig. 2e).

Bottom Line: When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled.In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force.This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.

View Article: PubMed Central - PubMed

Affiliation: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon 305-701, South Korea.

ABSTRACT
Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for membrane fusion, but its molecular mechanism is still unclear. Here using magnetic tweezers, we observe mechanical responses of a single neuronal SNARE complex under constant pulling force. Single SNARE complexes may be unzipped with 34 pN force. When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 11 pN. Thus, mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.

Show MeSH
Related in: MedlinePlus