Limits...
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.

Show MeSH
Mechanical hysteresis produces the partially assembled state of a single SNARE complex.(a–d) Unzipping and rezipping cycles of a single SNARE complex. A fully assembled SNARE complex at 11 pN (green traces) was unzipped by the application of a 34-pN force (red traces). When the force was lowered back to 11 pN (blue traces), the extension was higher than the corresponding extension observed before the 34-pN unzipping. This extension difference (D) disappeared when the force was removed (0 pN) and then restored to 11 pN (next green traces). The upper insets are the anticipated structures of the SNARE complex. The cylinders represent the α-helical structure, and the lines represent the unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) Distribution of the extension differences (D) with a Gaussian peak at 8.6±0.16 nm (s.e.m.) (N=63 force cycles from n=33 SNARE complexes). The s.d. is 2.79 nm. The percentages of the rezipping processes that stalled before the ionic layer or before the +2 layer were 66.7% and 87.3%, respectively. At the bottom, the estimated extensions of a single SNARE complex under an 11-pN force are shown. (f) Time behaviour of the extension differences normalized by their initial values, that is, D(t)/D(t=0), where t is the time spent in the second 11-pN phase (N=63 force cycles). In the statistical analyses of e and f, we included only data for SNARE complexes that showed repetitive unzipping and rezipping events. (g) A representative force-extension trace of a single SNARE complex shows unzipping above 30 pN and rezipping below 10 pN (average loading rate of 6 pN s−1 used).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3644077&req=5

f3: Mechanical hysteresis produces the partially assembled state of a single SNARE complex.(a–d) Unzipping and rezipping cycles of a single SNARE complex. A fully assembled SNARE complex at 11 pN (green traces) was unzipped by the application of a 34-pN force (red traces). When the force was lowered back to 11 pN (blue traces), the extension was higher than the corresponding extension observed before the 34-pN unzipping. This extension difference (D) disappeared when the force was removed (0 pN) and then restored to 11 pN (next green traces). The upper insets are the anticipated structures of the SNARE complex. The cylinders represent the α-helical structure, and the lines represent the unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) Distribution of the extension differences (D) with a Gaussian peak at 8.6±0.16 nm (s.e.m.) (N=63 force cycles from n=33 SNARE complexes). The s.d. is 2.79 nm. The percentages of the rezipping processes that stalled before the ionic layer or before the +2 layer were 66.7% and 87.3%, respectively. At the bottom, the estimated extensions of a single SNARE complex under an 11-pN force are shown. (f) Time behaviour of the extension differences normalized by their initial values, that is, D(t)/D(t=0), where t is the time spent in the second 11-pN phase (N=63 force cycles). In the statistical analyses of e and f, we included only data for SNARE complexes that showed repetitive unzipping and rezipping events. (g) A representative force-extension trace of a single SNARE complex shows unzipping above 30 pN and rezipping below 10 pN (average loading rate of 6 pN s−1 used).

Mentions: Figure 3 illustrates the full cycles of our constant-force application experiments. By applying the unzipping force of 34 pN, we first unzipped a fully assembled SNARE complex as in the experiments illustrated in Fig. 2 (Fig. 3a–d, red traces) and subsequently decreased the force back to 11 pN (blue traces) (hereafter, we use only the SNARE-DNA hybrid shown in Fig. 1 that shows C-to-N unzipping). Of note, the extension during this second 11-pN phase was higher than the extension during the first 11-pN phase before unzipping (Fig. 3a–d, blue versus green traces; see Supplementary Fig. S8 for more traces), suggesting that parts of the SNARE motifs remained disassembled. When we completely removed the tension (0-pN force), the extension was restored to the original value of the fully assembled SNARE complex, indicating complete rezipping of the SNARE complex. The SNARE-DNA hybrid system was then ready for another cycle of the experiment (Fig. 3a, arrows).


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)

Mechanical hysteresis produces the partially assembled state of a single SNARE complex.(a–d) Unzipping and rezipping cycles of a single SNARE complex. A fully assembled SNARE complex at 11 pN (green traces) was unzipped by the application of a 34-pN force (red traces). When the force was lowered back to 11 pN (blue traces), the extension was higher than the corresponding extension observed before the 34-pN unzipping. This extension difference (D) disappeared when the force was removed (0 pN) and then restored to 11 pN (next green traces). The upper insets are the anticipated structures of the SNARE complex. The cylinders represent the α-helical structure, and the lines represent the unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) Distribution of the extension differences (D) with a Gaussian peak at 8.6±0.16 nm (s.e.m.) (N=63 force cycles from n=33 SNARE complexes). The s.d. is 2.79 nm. The percentages of the rezipping processes that stalled before the ionic layer or before the +2 layer were 66.7% and 87.3%, respectively. At the bottom, the estimated extensions of a single SNARE complex under an 11-pN force are shown. (f) Time behaviour of the extension differences normalized by their initial values, that is, D(t)/D(t=0), where t is the time spent in the second 11-pN phase (N=63 force cycles). In the statistical analyses of e and f, we included only data for SNARE complexes that showed repetitive unzipping and rezipping events. (g) A representative force-extension trace of a single SNARE complex shows unzipping above 30 pN and rezipping below 10 pN (average loading rate of 6 pN s−1 used).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Mechanical hysteresis produces the partially assembled state of a single SNARE complex.(a–d) Unzipping and rezipping cycles of a single SNARE complex. A fully assembled SNARE complex at 11 pN (green traces) was unzipped by the application of a 34-pN force (red traces). When the force was lowered back to 11 pN (blue traces), the extension was higher than the corresponding extension observed before the 34-pN unzipping. This extension difference (D) disappeared when the force was removed (0 pN) and then restored to 11 pN (next green traces). The upper insets are the anticipated structures of the SNARE complex. The cylinders represent the α-helical structure, and the lines represent the unstructured regions (blue: synaptobrevin 2, red: syntaxin 1A, light yellow: SNAP-25). (e) Distribution of the extension differences (D) with a Gaussian peak at 8.6±0.16 nm (s.e.m.) (N=63 force cycles from n=33 SNARE complexes). The s.d. is 2.79 nm. The percentages of the rezipping processes that stalled before the ionic layer or before the +2 layer were 66.7% and 87.3%, respectively. At the bottom, the estimated extensions of a single SNARE complex under an 11-pN force are shown. (f) Time behaviour of the extension differences normalized by their initial values, that is, D(t)/D(t=0), where t is the time spent in the second 11-pN phase (N=63 force cycles). In the statistical analyses of e and f, we included only data for SNARE complexes that showed repetitive unzipping and rezipping events. (g) A representative force-extension trace of a single SNARE complex shows unzipping above 30 pN and rezipping below 10 pN (average loading rate of 6 pN s−1 used).
Mentions: Figure 3 illustrates the full cycles of our constant-force application experiments. By applying the unzipping force of 34 pN, we first unzipped a fully assembled SNARE complex as in the experiments illustrated in Fig. 2 (Fig. 3a–d, red traces) and subsequently decreased the force back to 11 pN (blue traces) (hereafter, we use only the SNARE-DNA hybrid shown in Fig. 1 that shows C-to-N unzipping). Of note, the extension during this second 11-pN phase was higher than the extension during the first 11-pN phase before unzipping (Fig. 3a–d, blue versus green traces; see Supplementary Fig. S8 for more traces), suggesting that parts of the SNARE motifs remained disassembled. When we completely removed the tension (0-pN force), the extension was restored to the original value of the fully assembled SNARE complex, indicating complete rezipping of the SNARE complex. The SNARE-DNA hybrid system was then ready for another cycle of the experiment (Fig. 3a, arrows).

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