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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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Zippering of the C-terminal half of a single SNARE complex.(a–d) Force-cycle experiments for studying the C-terminal-half zippering of single SNARE complexes. By unzipping a single SNARE complex at 34 pN and subsequently lowering the force to 11 pN (a), 9.1 pN (b), 7.5 pN (c) and 5.2 pN (d), the SNARE complex can be trapped in its partially assembled state (blue traces). Then, during the observation time of 20 s, the extension difference is monitored to observe the occurrences of the C-terminal-half zippering, as indicated by the restoration of the original extension (changes from blue to green traces in c and d). The fraction of partially assembled states that do not show such C-terminal zipping within the observation time is defined as the survival probability. (e) Survival probability after 20 s of observation () as a function of pulling force (N=27, 23, 31 and 26 events from n=15, 11, 10 and 11 SNARE complexes for 11, 9.1, 7.5 and 5.2 pN, respectively). (f) Fitting the force dependence of the survival probability with equation (1) gives =2.93 nm and =5.93 s−1, where  is the position of the energy barrier confining the partially assembled state and  is the kinetic rate for zippering of the C-terminal half at zero force.
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f4: Zippering of the C-terminal half of a single SNARE complex.(a–d) Force-cycle experiments for studying the C-terminal-half zippering of single SNARE complexes. By unzipping a single SNARE complex at 34 pN and subsequently lowering the force to 11 pN (a), 9.1 pN (b), 7.5 pN (c) and 5.2 pN (d), the SNARE complex can be trapped in its partially assembled state (blue traces). Then, during the observation time of 20 s, the extension difference is monitored to observe the occurrences of the C-terminal-half zippering, as indicated by the restoration of the original extension (changes from blue to green traces in c and d). The fraction of partially assembled states that do not show such C-terminal zipping within the observation time is defined as the survival probability. (e) Survival probability after 20 s of observation () as a function of pulling force (N=27, 23, 31 and 26 events from n=15, 11, 10 and 11 SNARE complexes for 11, 9.1, 7.5 and 5.2 pN, respectively). (f) Fitting the force dependence of the survival probability with equation (1) gives =2.93 nm and =5.93 s−1, where is the position of the energy barrier confining the partially assembled state and is the kinetic rate for zippering of the C-terminal half at zero force.

Mentions: Finally, when the force was lowered by only a few pN from this stably-clamping 11-pN force, we observed the assembly of the C-terminal parts to complete the SNARE complex structure (Fig. 4). To quantitatively characterize the SNARE-complex assembly from the partially assembled state, we repeated the force cycle experiment shown in Fig. 3, but decreased the initial and final force levels from the original 11 pN to 9.1, 7.5 and 5.2 pN (Fig. 4a–d). The assembly of the C-terminal part was detected by a decrease in the extension, as confirmed by the restoration of the original extension of the fully assembled SNARE complex (Fig. 4c, change from blue to green traces).


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)

Zippering of the C-terminal half of a single SNARE complex.(a–d) Force-cycle experiments for studying the C-terminal-half zippering of single SNARE complexes. By unzipping a single SNARE complex at 34 pN and subsequently lowering the force to 11 pN (a), 9.1 pN (b), 7.5 pN (c) and 5.2 pN (d), the SNARE complex can be trapped in its partially assembled state (blue traces). Then, during the observation time of 20 s, the extension difference is monitored to observe the occurrences of the C-terminal-half zippering, as indicated by the restoration of the original extension (changes from blue to green traces in c and d). The fraction of partially assembled states that do not show such C-terminal zipping within the observation time is defined as the survival probability. (e) Survival probability after 20 s of observation () as a function of pulling force (N=27, 23, 31 and 26 events from n=15, 11, 10 and 11 SNARE complexes for 11, 9.1, 7.5 and 5.2 pN, respectively). (f) Fitting the force dependence of the survival probability with equation (1) gives =2.93 nm and =5.93 s−1, where  is the position of the energy barrier confining the partially assembled state and  is the kinetic rate for zippering of the C-terminal half at zero force.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Zippering of the C-terminal half of a single SNARE complex.(a–d) Force-cycle experiments for studying the C-terminal-half zippering of single SNARE complexes. By unzipping a single SNARE complex at 34 pN and subsequently lowering the force to 11 pN (a), 9.1 pN (b), 7.5 pN (c) and 5.2 pN (d), the SNARE complex can be trapped in its partially assembled state (blue traces). Then, during the observation time of 20 s, the extension difference is monitored to observe the occurrences of the C-terminal-half zippering, as indicated by the restoration of the original extension (changes from blue to green traces in c and d). The fraction of partially assembled states that do not show such C-terminal zipping within the observation time is defined as the survival probability. (e) Survival probability after 20 s of observation () as a function of pulling force (N=27, 23, 31 and 26 events from n=15, 11, 10 and 11 SNARE complexes for 11, 9.1, 7.5 and 5.2 pN, respectively). (f) Fitting the force dependence of the survival probability with equation (1) gives =2.93 nm and =5.93 s−1, where is the position of the energy barrier confining the partially assembled state and is the kinetic rate for zippering of the C-terminal half at zero force.
Mentions: Finally, when the force was lowered by only a few pN from this stably-clamping 11-pN force, we observed the assembly of the C-terminal parts to complete the SNARE complex structure (Fig. 4). To quantitatively characterize the SNARE-complex assembly from the partially assembled state, we repeated the force cycle experiment shown in Fig. 3, but decreased the initial and final force levels from the original 11 pN to 9.1, 7.5 and 5.2 pN (Fig. 4a–d). The assembly of the C-terminal part was detected by a decrease in the extension, as confirmed by the restoration of the original extension of the fully assembled SNARE complex (Fig. 4c, change from blue to green traces).

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