We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a chi2 near unity. The chi2 surface suggests an ultimate level of precision with FRET of < 1% of the Förster radius. Distributions of FRET signals in donor-acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 A inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the Förster radius are ruled out.

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.

We hypothesize that these homo- and heteromeric complexes may differentially regulate distal root meristem maintenance. We conclude that essential components of the ancestral shoot stemness regulatory system also act in the root and that the specific interaction of CLV1 with ACR4 serves to moderate and control stemness homeostasis in the root meristem. The structural differences between these two meristem types may have necessitated this recruitment of ACR4 for signaling by CLV1.

By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.H IV-1 reverse transcriptase (RT) is a key enzyme in the life cycle of HIV. This multifunctional enzyme is responsible for the complex process of transcribing viral RNA into doublestranded DNA for integration into the host cell genome. The enzyme is a heterodimer composed of subunits which share a common N terminus and have subdomains referred to as fingers, palm, thumb, and connection ( Fig. 1). Although the subdomains of each subunit are structurally similar, the overall folding within the two subunits is quite different (1). The large subunit, p66, contains RNA-and DNA-dependent DNA polymerase as well as RNase H activities. The p51 subunit, which is inactive in the heterodimer but active in homodimers (2, 3), is thought to play a role in stabilizing the conformation of p66.Crystallographic studies of RT:nucleic acid primer͞template (p͞t) complexes have provided valuable insights into the structural features and conformational changes induced by p͞t binding (1, 4-7). To date, these crystallographic models have indicated a single p͞t-binding mode. In contrast, recent solutionbased kinetic studies on p͞t binding and nucleotide incorporation by RT suggest a heterogeneous mixture of several different binding modes (8). To confirm directly the existence of several species, and to obtain structural and functional information about each, we have used single-molecule spectroscopy to investigate RT:p͞t complexes in solution.Single-molecule techniques have proven to be valuable tools for resolving static and dynamic heterogeneity of an ensemble (9-13). For this investigation, we use a newly developed multiparameter fluorescence detection (MF...

Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Forster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied to characterize conformational exchange between two or more states via global analysis of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quantitatively recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time t(d) (typically milliseconds), with a dynamic range of the method of about +/-1 order of magnitude with respect to t(d). Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimolecular events. Second, an analysis procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a maximum likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Analysis tools for direct visual inspection of two-dimensional diagrams of FRET efficiency versus donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated ''Widom 601'' positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.chromatin ͉ mononucleosomes ͉ structure T he nucleosome, the basic unit of genome compaction (1), consists of Ϸ2 turns of double-stranded DNA wound around a histone protein octamer. Its structure is known to a resolution of 1.9 Å (2). DNA sequence, chemical modifications and histone composition modulate gene activity through this structure.Central to nucleosomal function is its restructuring during processes that act on DNA, e.g., transcription or replication. Biochemical data indicate that nucleosome dissociation is temporary. Mechanisms for nucleosome unfolding, unwrapping or repositioning have been proposed (3), but so far none has been proven by direct physical evidence (e.g., detection of intermediate states). Here, we use single molecule Förster resonance energy transfer (FRET) to obtain quantitative structural information for elucidating such mechanisms.Bulk solution FRET is a proven tool for measuring average distances; nucleosome dynamics have been quantified by FRET between dyes attached to DNA and/or histone proteins (4-6). Analyzing FRET from single molecules (7, 8) can give detailed information on structural diversity; e.g., FRET on surfacetethered nucleosomes demonstrated spontaneous structure fluctuations (9, 10), whereas confocal spFRET experiments (11, 12) on freely diffusing single nucleosomes detected structural subpopulations under various conditions.The transit of a single molecule through the laser focus ...

GBPs are essential for immunity against intracellular pathogens, especially for Toxoplasma gondii control. Here, the molecular interactions of murine GBPs (mGBP1/2/3/5/6), homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, and live cell microscopy methodologies. Control of T. gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus, enabling reversible oligomerization in vesicle-like structures. mGBP2 undergoes structural transitions between monomeric, dimeric and oligomeric states visualized by quantitative FRET analysis. mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. This dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity.DOI: http://dx.doi.org/10.7554/eLife.11479.001