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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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Model for synaptic vesicle fusion based on our mechanical study of single SNARE complexes.(a–d) Model for synaptic vesicle fusion. In the initial phase of synaptic vesicle fusion, the two membranes are far apart and the repulsion force on a single SNARE complex should be negligible (a). When the effective repulsion on a single SNARE complex is larger than 11 pN (but smaller than 34 pN), the SNARE complex becomes trapped in the partially assembled state, in which the C-terminal half of the SNARE motif is selectively disassembled (b). Zippering of the remaining C-terminal half can be triggered by the work of fusion regulators such as synaptotagmin (c) and/or the formation of additional SNARE complexes (d). (e) Energy landscape diagrams for the SNARE-complex formation at 0-pN and 11-pN forces. (f) Energy barriers separating the partially assembled state from the fully assembled state of a single SNARE complex. The parameters of the energy barrier, ,  and , are shown for various force values.
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f5: Model for synaptic vesicle fusion based on our mechanical study of single SNARE complexes.(a–d) Model for synaptic vesicle fusion. In the initial phase of synaptic vesicle fusion, the two membranes are far apart and the repulsion force on a single SNARE complex should be negligible (a). When the effective repulsion on a single SNARE complex is larger than 11 pN (but smaller than 34 pN), the SNARE complex becomes trapped in the partially assembled state, in which the C-terminal half of the SNARE motif is selectively disassembled (b). Zippering of the remaining C-terminal half can be triggered by the work of fusion regulators such as synaptotagmin (c) and/or the formation of additional SNARE complexes (d). (e) Energy landscape diagrams for the SNARE-complex formation at 0-pN and 11-pN forces. (f) Energy barriers separating the partially assembled state from the fully assembled state of a single SNARE complex. The parameters of the energy barrier, , and , are shown for various force values.

Mentions: Our results collectively suggest that the process of synaptic vesicle fusion is a delicate function of the repulsion force between the two fusing membranes (Fig. 5). In the initial phase of membrane fusion, the two membranes are far apart, and the repulsion force on a single SNARE complex should be negligible (Fig. 5a). Our mechanical unzipping of single SNARE complexes reveals that the middle layers around the ionic layer are important for the mechanical stability of an individual SNARE complex. This result is consistent with the fact that the sequence of these middle layers, between the −2 and +2 layers, faithfully follows the leucine zipper model3738. A fusion process would practically commence when those centre layers assemble to form a minimal, stable SNARE complex. With this SNARE complex formation, the repulsion force should steeply increase as the two fusing membranes are brought to nm-scale apposition. Most importantly, our results collectively suggest that if the effective repulsion force on a single SNARE complex becomes larger than 11 pN (but smaller than 34 pN), each SNARE complex will be trapped in the ‘partially assembled’ state without requiring any help from other auxiliary proteins (Fig. 5b).


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)

Model for synaptic vesicle fusion based on our mechanical study of single SNARE complexes.(a–d) Model for synaptic vesicle fusion. In the initial phase of synaptic vesicle fusion, the two membranes are far apart and the repulsion force on a single SNARE complex should be negligible (a). When the effective repulsion on a single SNARE complex is larger than 11 pN (but smaller than 34 pN), the SNARE complex becomes trapped in the partially assembled state, in which the C-terminal half of the SNARE motif is selectively disassembled (b). Zippering of the remaining C-terminal half can be triggered by the work of fusion regulators such as synaptotagmin (c) and/or the formation of additional SNARE complexes (d). (e) Energy landscape diagrams for the SNARE-complex formation at 0-pN and 11-pN forces. (f) Energy barriers separating the partially assembled state from the fully assembled state of a single SNARE complex. The parameters of the energy barrier, ,  and , are shown for various force values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3644077&req=5

f5: Model for synaptic vesicle fusion based on our mechanical study of single SNARE complexes.(a–d) Model for synaptic vesicle fusion. In the initial phase of synaptic vesicle fusion, the two membranes are far apart and the repulsion force on a single SNARE complex should be negligible (a). When the effective repulsion on a single SNARE complex is larger than 11 pN (but smaller than 34 pN), the SNARE complex becomes trapped in the partially assembled state, in which the C-terminal half of the SNARE motif is selectively disassembled (b). Zippering of the remaining C-terminal half can be triggered by the work of fusion regulators such as synaptotagmin (c) and/or the formation of additional SNARE complexes (d). (e) Energy landscape diagrams for the SNARE-complex formation at 0-pN and 11-pN forces. (f) Energy barriers separating the partially assembled state from the fully assembled state of a single SNARE complex. The parameters of the energy barrier, , and , are shown for various force values.
Mentions: Our results collectively suggest that the process of synaptic vesicle fusion is a delicate function of the repulsion force between the two fusing membranes (Fig. 5). In the initial phase of membrane fusion, the two membranes are far apart, and the repulsion force on a single SNARE complex should be negligible (Fig. 5a). Our mechanical unzipping of single SNARE complexes reveals that the middle layers around the ionic layer are important for the mechanical stability of an individual SNARE complex. This result is consistent with the fact that the sequence of these middle layers, between the −2 and +2 layers, faithfully follows the leucine zipper model3738. A fusion process would practically commence when those centre layers assemble to form a minimal, stable SNARE complex. With this SNARE complex formation, the repulsion force should steeply increase as the two fusing membranes are brought to nm-scale apposition. Most importantly, our results collectively suggest that if the effective repulsion force on a single SNARE complex becomes larger than 11 pN (but smaller than 34 pN), each SNARE complex will be trapped in the ‘partially assembled’ state without requiring any help from other auxiliary proteins (Fig. 5b).

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