Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.

Mutations in Membrin/GOSR2reveal stringent secretory pathway demands of dendritic growth and synaptic integrity

“…The magnitude of these effects is consistent with the position and nature of each mutation within the SNARE motif. The D196del deletion likely results in misalignment of the subsequent hydrophobic layers in the C-terminal half of the SNARE domain, a region that provides the critical force to drive membrane fusion (Zhang, 2017). Correspondingly, this mutation reduced total fusion (after 120 min) by ~ 60% ( Figure 1C, D).…”

Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly

“…2B). However, since these transitions occur during constant pulling, some slow transitions may not reach thermodynamic equilibrium, leading to hysteresis in the force extension curves (16, 33). …”

“…It is challenging to observe multiple partially assembled SNARE intermediates and measure the force generated by SNARE assembly using traditional experimental approaches (33, 34). As a result, much of our knowledge on regulated SNARE assembly is only inferred from interactions of regulator proteins with fully assembled SNARE complexes or readouts of membrane fusion (35–40).…”

“…As a result, much of our knowledge on regulated SNARE assembly is only inferred from interactions of regulator proteins with fully assembled SNARE complexes or readouts of membrane fusion (35–40). The single-molecule manipulation approach based on high-resolution optical tweezers is one of a few methods capable of measuring both the structural intermediates of SNARE assembly and their associated forces, energies, and kinetics (15, 16, 33, 41–43). Here, we describe our detailed protocols to characterize SNARE assembly regulated by α-SNAP (42).…”

Editorial Overview: Single-Molecule Approaches to Difficult Challenges in Folding and Dynamics

“…Using high-resolution optical tweezers, Rebane et al investigated coupled folding and assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) [20]. Unlike most proteins whose folding precedes their biological functions, SNARE folding per se serves as their major function, which is coupled to synaptic vesicle fusion and neurotransmitter release [21]. As a reaction product, the fully assembly SNARE complex needs to be disassembled and recycled for next round of membrane fusion.…”