Structure of Synaptic Membrane Fusion Protein

The SNARE complex is known as a ‘fusion machine’ that generates the mechanical force, which is necessary to overcome the free energy barrier for fusion of two membranes. However, the force that a single SNARE complex generates and the mechanics of it are largely unknown because of the experimental difficulties in pulling a single SNARE complex apart mechanically.

We used magnetic tweezers techniques to monitor the unzipping and re-zipping processes of a single SNARE complex under constant-force conditions. This experiment reveals unprecedented details of the mechanics of how the single SNARE fusion machine might work in response to the bilayer repulsion force. First, through unzipping the SNARE core by magnetic tweezers, we’ve learned that it requires as much as 35 pN to dissociate synaptobrevin 2 from the SNARE complex and we often observe a clear two step unzipping, suggesting that SNARE complex formation occurs in two steps. The breaking point is estimated to be near the highly conserved ionic zero layer. Second, during the course of rezipping, we have discovered that rezipping can be held off with mere 11 pN, which is more than 20 pN less than the force required for unzipping. In this highly metastable state due to the mechanical hysteresis, the C-terminal half of the SNARE motifs stably remains unzipped, which marks the first direction observation of ‘the partially-assembled state’ of the neuronal SNARE complex. When the force is reduced by only a few pN from this stably-clamping 11 pN, the assembly of the C-terminal half can be triggered to complete formation of a single SNARE complex.

• Mechanical unzipping and rezipping of a single SNARE complex reveals large hysteresis as force-generating mechanism, Nature Communications 4, 1705 (2013).

Real-Time Single-Molecule Co-immuoprecipitation

Co-immunoprecipitation (Co-IP) is an immunoprecipitation of intact protein complexes, which works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. We developed the real-time single-molecule co-immuoprecipitation (co-IP) analysis, which provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, it is possible to determine the signaling kinetics of native proteins and detect changes in the protein-protein interactions in a given tumor tissue. The detailed protocol for the real-time single-molecule co-IP analysis was published to Nature Protocols. In the protocols paper, we also provided a total solution for computer programs, named SMET (Single-Molecule Experiment Tools) which enables the users to perform all the steps of the real-time single-molecule co-IP analysis, from real-time single-molecule imaging to final extraction of kinetic rates. Step-by-step experimental protocols are provided along with the detailed instructions on how to use the SMET programs for each step.

• Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions, Nature Protocols 8, 2045 (2013).