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...

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

In this study, we demonstrate two new concepts, using fluorescence correlation spectroscopy (FCS), to characterize fluorescence resonance energy transfer (FRET). The two approaches were tested experimentally by measuring a series of double-stranded DNA molecules, with different numbers of base-pairs separating the donor (Alexa488) and acceptor (Cy5) fluorophores. In the first approach, FRET efficiencies are determined from the detected acceptor fluorescence rate per molecule. Here, the unique possibility with FCS to determine the mean number of molecules within the detection volume is exploited, making a concentration calibration superfluous. The second approach takes advantage of FRET-dependent fluorescence fluctuations of photophysical origin, in particular fluctuations generated by trans−cis isomerization of the acceptor dye. The rate of interchange between the trans and cis states is proportional to the excitation rate and can be conveniently measured by FCS. Under FRET-mediated excitation, this rate can be used as a direct measure of the FRET efficiency. The measured isomerization rate depends only on the fluctuations in the acceptor fluorescence, and is not affected by donor fluorescence cross-talk, background, dye labeling efficiencies, or by the concentration of molecules under study. The measured FRET efficiencies are well in agreement with a structural model of DNA. Furthermore, additional structural information is obtained from simulations of the measured fraction of acceptor dyes being in a nonfluorescent cis conformation, from which differences in the position and orientation of the trans and cis form of the acceptor dye can be predicted.

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2-Aza-2'-deoxyadenosine (2, z 2 A d ) is synthesized via its 1,N 6 -etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10 b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c 7 A d ), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.

2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.

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