Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1

Abstract

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins have emerged as the leading candidates for mediating membrane fusion. They comprise a superfamily of small membrane proteins distinguished by the SNARE motif, a conserved coiled-coil stretch of 60-70 amino acids. SNARE motifs spontaneously assemble into elongated four-helix bundles in which each helix is contributed by a SNARE motif belonging to a separate subclass. Complex formation is assumed to tie membranes together and to initiate membrane fusion along a reaction path involving so-farunknown conformational transitions (1-3).In most SNAREs, the SNARE motif is located adjacent to a C-terminal transmembrane domain. Furthermore, many SNAREs contain an independently folded domain at the N terminus that is connected to the SNARE motif by a linker region. In the syntaxin subfamily (also referred to as QaSNAREs), the N-terminal domains consist of antiparallel bundles of three ␣-helices that are structurally conserved despite high divergence in the primary structure. The N-terminal domains of several syntaxins interact reversibly with the SNARE motif, resulting in two distinct conformations; a closed conformation in which the SNARE motif is blocked (i.e., unable to form SNARE complexes), and an open conformation in which there is presumably no contact between these domains (2). Binding of munc-18, a regulatory protein essential for exocytosis, arrests syntaxin 1 in the closed conformation in which the N-terminal portion of the SNARE motif binds to a groove on the surface of the Habc domain (ref. 4 and Fig. 1). Mutations destabilizing the closed state of syntaxin have profound effects on exocytosis, suggesting that the conformational transition is a key element in the biological function of syntaxin 1 (4, 5).Conformational transitions such as those discussed above are difficult to observe directly due to limited temporal or spatial resolution. To overcome these limitations, we have recently developed a si...

Determinants of Synaptobrevin Regulation in Membranes

“…As reported for the soluble form of synaptobrevin (Fasshauer and Margittai, 2004), the reaction rate was accelerated by increasing the concentration of SNAP-25 (Supplementary Figure S4). Conversely, the rate is reduced when the entire cytoplasmic region of syntaxin is used (Supplementary Figure S2), in agreement with the notion that the N-terminal regulatory Habc-domain slows down SNARE assembly (Margittai et al, 2003). Furthermore, addition of detergent to synaptobrevincontaining liposomes led to only moderate acceleration of the reaction rate (Supplementary Figure S5), suggesting that there is no significant difference in reactivity regardless of whether synaptobrevin is anchored to bilayers or inserted in detergent micelles.…”

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“…22,23 In general, however, the number of photons in a single burst is usually insufficient to perform any time-trace analysis, except in a few rare cases of long bursts. 24 Instead, in smFRET diffusion experiments, quantities are computed at the burst level using all photons in each burst and then histogrammed.…”

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…104 The result was a clearly bimodal distribution indicating fluctuations between two distinct states within bursts. Occasionally, some rare bursts can last several milliseconds, during which fluctuations can possibly be observed directly.…”