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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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Magnetic tweezers for studying the mechanical behaviour of a single neuronal SNARE complex.(a) Experimental scheme. The pulling force is generated by bringing a pair of magnets near a magnetic bead and is delivered to a single SNARE complex through DNA handles. The neuronal SNARE complex is composed of synaptobrevin 2 (blue coil), syntaxin 1A (red coil) and SNAP-25 (light yellow coils). (b) Detailed structure of the SNARE-DNA hybrid. The layered structure of the SNARE complex and the positions of the DNA handles and the internal disulphide bond are shown. The wheel diagrams of the +7 and −7 layers are shown, with the positions of the heptad repeats indicated by letters from a to g. (c) Representative force-extension trace of a single SNARE complex. An average loading rate of 1 pN s−1 was used. The trace shows two sequential unzipping events (black arrows), and the inset shows the whole trace covering the force range from 0 to 50 pN. (d) Preliminary force-jump experiments. The pulling force was increased from 11–34 pN with three different loading rates of 1, 8 and 58 pN s−1, and was subsequently maintained at a constant force of 34 pN. The dashed line indicates the time when the force reaches 34 pN, and the arrow indicates unzipping events. The time lapse (Δt) was measured from the dashed line to the arrow in an individual trace. (e) Average Δt as a function of loading rate. The numbers of events (N) are 18, 15 and 119, and the numbers of SNARE complexes used (n) are 12, 7 and 41 for 1, 8 and 58 pN s−1, respectively. dsDNA, double-stranded DNA.
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f1: Magnetic tweezers for studying the mechanical behaviour of a single neuronal SNARE complex.(a) Experimental scheme. The pulling force is generated by bringing a pair of magnets near a magnetic bead and is delivered to a single SNARE complex through DNA handles. The neuronal SNARE complex is composed of synaptobrevin 2 (blue coil), syntaxin 1A (red coil) and SNAP-25 (light yellow coils). (b) Detailed structure of the SNARE-DNA hybrid. The layered structure of the SNARE complex and the positions of the DNA handles and the internal disulphide bond are shown. The wheel diagrams of the +7 and −7 layers are shown, with the positions of the heptad repeats indicated by letters from a to g. (c) Representative force-extension trace of a single SNARE complex. An average loading rate of 1 pN s−1 was used. The trace shows two sequential unzipping events (black arrows), and the inset shows the whole trace covering the force range from 0 to 50 pN. (d) Preliminary force-jump experiments. The pulling force was increased from 11–34 pN with three different loading rates of 1, 8 and 58 pN s−1, and was subsequently maintained at a constant force of 34 pN. The dashed line indicates the time when the force reaches 34 pN, and the arrow indicates unzipping events. The time lapse (Δt) was measured from the dashed line to the arrow in an individual trace. (e) Average Δt as a function of loading rate. The numbers of events (N) are 18, 15 and 119, and the numbers of SNARE complexes used (n) are 12, 7 and 41 for 1, 8 and 58 pN s−1, respectively. dsDNA, double-stranded DNA.

Mentions: To probe the mechanical response of a single SNARE complex, we constructed a SNARE-DNA hybrid, in which 510 base-pair double-stranded DNAs were attached to the C-terminal ends (right after the +7 layer) of syntaxin 1 A and synaptobrevin 2 using a thiol-disulphide exchange reaction272829 (Fig. 1a and Supplementary Fig. S2). Specifically, the DNA handles were attached to the exterior surface of the SNARE complex to minimize any perturbation to the formation of the four-helix bundle. We removed the Habc domain of syntaxin 1A and the transmembrane domains, but the entire SNARE motifs and linker regions of syntaxin 1A (amino acids 191–266) and synaptobrevin 2 (1–94) were included in our experiments (Methods and Supplementary Fig. S3). The −7 layer near the N-terminal end of the SNARE complex was covalently knotted by a cysteine-bridge for repetitive unzipping and rezipping experiments for a single molecule28 (Fig. 1b). As the formation of the SNARE complex is thought to initiate in the N-terminal region and propagate toward the C-terminal ends, our positions for the DNA handles and cysteine-bridge knotting were chosen to induce unzipping in the C- to N-terminal direction and rezipping of a single SNARE complex in the opposite, N-to C-terminal direction. Thus, this rezipping process is designed to closely mimic the native zippering direction of the SNARE complex. This SNARE-DNA hybrid was connected to a polyethylene glycol-coated cover glass and a magnetic bead through biotin-neutravidin and dig-antidig linkages, respectively.


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)

Magnetic tweezers for studying the mechanical behaviour of a single neuronal SNARE complex.(a) Experimental scheme. The pulling force is generated by bringing a pair of magnets near a magnetic bead and is delivered to a single SNARE complex through DNA handles. The neuronal SNARE complex is composed of synaptobrevin 2 (blue coil), syntaxin 1A (red coil) and SNAP-25 (light yellow coils). (b) Detailed structure of the SNARE-DNA hybrid. The layered structure of the SNARE complex and the positions of the DNA handles and the internal disulphide bond are shown. The wheel diagrams of the +7 and −7 layers are shown, with the positions of the heptad repeats indicated by letters from a to g. (c) Representative force-extension trace of a single SNARE complex. An average loading rate of 1 pN s−1 was used. The trace shows two sequential unzipping events (black arrows), and the inset shows the whole trace covering the force range from 0 to 50 pN. (d) Preliminary force-jump experiments. The pulling force was increased from 11–34 pN with three different loading rates of 1, 8 and 58 pN s−1, and was subsequently maintained at a constant force of 34 pN. The dashed line indicates the time when the force reaches 34 pN, and the arrow indicates unzipping events. The time lapse (Δt) was measured from the dashed line to the arrow in an individual trace. (e) Average Δt as a function of loading rate. The numbers of events (N) are 18, 15 and 119, and the numbers of SNARE complexes used (n) are 12, 7 and 41 for 1, 8 and 58 pN s−1, respectively. dsDNA, double-stranded DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3644077&req=5

f1: Magnetic tweezers for studying the mechanical behaviour of a single neuronal SNARE complex.(a) Experimental scheme. The pulling force is generated by bringing a pair of magnets near a magnetic bead and is delivered to a single SNARE complex through DNA handles. The neuronal SNARE complex is composed of synaptobrevin 2 (blue coil), syntaxin 1A (red coil) and SNAP-25 (light yellow coils). (b) Detailed structure of the SNARE-DNA hybrid. The layered structure of the SNARE complex and the positions of the DNA handles and the internal disulphide bond are shown. The wheel diagrams of the +7 and −7 layers are shown, with the positions of the heptad repeats indicated by letters from a to g. (c) Representative force-extension trace of a single SNARE complex. An average loading rate of 1 pN s−1 was used. The trace shows two sequential unzipping events (black arrows), and the inset shows the whole trace covering the force range from 0 to 50 pN. (d) Preliminary force-jump experiments. The pulling force was increased from 11–34 pN with three different loading rates of 1, 8 and 58 pN s−1, and was subsequently maintained at a constant force of 34 pN. The dashed line indicates the time when the force reaches 34 pN, and the arrow indicates unzipping events. The time lapse (Δt) was measured from the dashed line to the arrow in an individual trace. (e) Average Δt as a function of loading rate. The numbers of events (N) are 18, 15 and 119, and the numbers of SNARE complexes used (n) are 12, 7 and 41 for 1, 8 and 58 pN s−1, respectively. dsDNA, double-stranded DNA.
Mentions: To probe the mechanical response of a single SNARE complex, we constructed a SNARE-DNA hybrid, in which 510 base-pair double-stranded DNAs were attached to the C-terminal ends (right after the +7 layer) of syntaxin 1 A and synaptobrevin 2 using a thiol-disulphide exchange reaction272829 (Fig. 1a and Supplementary Fig. S2). Specifically, the DNA handles were attached to the exterior surface of the SNARE complex to minimize any perturbation to the formation of the four-helix bundle. We removed the Habc domain of syntaxin 1A and the transmembrane domains, but the entire SNARE motifs and linker regions of syntaxin 1A (amino acids 191–266) and synaptobrevin 2 (1–94) were included in our experiments (Methods and Supplementary Fig. S3). The −7 layer near the N-terminal end of the SNARE complex was covalently knotted by a cysteine-bridge for repetitive unzipping and rezipping experiments for a single molecule28 (Fig. 1b). As the formation of the SNARE complex is thought to initiate in the N-terminal region and propagate toward the C-terminal ends, our positions for the DNA handles and cysteine-bridge knotting were chosen to induce unzipping in the C- to N-terminal direction and rezipping of a single SNARE complex in the opposite, N-to C-terminal direction. Thus, this rezipping process is designed to closely mimic the native zippering direction of the SNARE complex. This SNARE-DNA hybrid was connected to a polyethylene glycol-coated cover glass and a magnetic bead through biotin-neutravidin and dig-antidig linkages, respectively.

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