Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

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Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1, a synaptic vesicle protein that interacts with SNAREs and membranes. We used single-molecule FRET between synaptotagmin’s two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intra-synaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive synaptotagmin 1–SNARE complex by using a multi-body docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they could interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+ bound synaptotagmin 3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs plays a role in Ca2+-triggered fusion.

A single molecule fluorescence assay is presented for studying the mechanism of soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs)-mediated liposome fusion to supported lipid bilayers. The three neuronal SNAREs syntaxin-1A, synaptobrevin-II (VAMP), and SNAP-25A were expressed separately, and various dye-labeled combinations of the SNAREs were tested for their ability to dock liposomes and induce fusion. Syntaxin and synaptobrevin in opposing membranes were both necessary and sufficient to dock liposomes to supported bilayers and to induce thermally activated fusion. As little as one SNARE interaction was sufficient for liposome docking. Fusion of docked liposomes with the supported bilayer was monitored by the dequenching of soluble fluorophores entrapped within the liposomes. Fusion was stimulated by illumination with laser light, and the fusion probability was enhanced by raising the ambient temperature from 22 to 37 degrees C, suggesting a thermally activated process. Surprisingly, SNAP-25 had little effect on docking efficiency or the probability of thermally induced fusion. Interprotein fluorescence resonance energy transfer experiments suggest the presence of other conformational states of the syntaxin*synaptobrevin interaction in addition to those observed in the crystal structure of the SNARE complex. Furthermore, although SNARE complexes involved in liposome docking preferentially assemble into a parallel configuration, both parallel and antiparallel configurations were observed.

Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.