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.

Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h ^–1 . The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N -ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo , and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro .

SNAREs are essential components of the machinery for Ca²⁺-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca²⁺ sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.