Single-Molecule Study of Neuronal t-SNARE Structure, Stability and Dynamics Using Optical Tweezers

Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) couple their stepwise folding to membrane fusion. For numerous SNAREs, the first and rate-limiting step of their folding is the assembly of t-SNAREs into a binary complex. However, the structure, stability, and dynamics of this t-SNARE complex remain elusive. Here we show that the neuronal t-SNARE complex is a three-helix bundle with a frayed C-terminus using a single-molecule manipulation approach based on high-resolution optical tweezers. The helical bundle contains two sequentially folding domains: an N-terminal domain (NTD) and a C-terminal domain (CTD). A central ionic layer separates the two domains and plays a critical role in t-SNARE folding. Point mutation of the ionic layer alters the folding pathway and structure of the t-SNARE complex. The folding of the wild-type t-SNARE complex releases a total free energy of ~10.5 kBT, with ~4.8 kBT in the NTD and ~5.7 kBT in the CTD. The binding of v-SNARE VAMP-2 to the NTD of the t-SNARE complex stabilizes the CTD of the complex, thereby facilitating rapid zippering at the CTD during membrane fusion. Our results support the SNARE zippering model and provide a molecular mechanism for the hypothesis that t-SNAREs act as a zippering template for v-SNARE during membrane fusion.