Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCRtargeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β 2 -adrenergic receptor (β 2 AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.signal transduction mechanisms | agonists and inverse agonists | conformational polymorphism | single-molecule fluorescence spectroscopy | phospholipid nanodiscs G protein-coupled receptors (GPCRs) mediate a multitude of physiological functions and are the targets for a myriad of drugs (1), many of which elicit different functional outcomes through the same receptor (2). It remains to be rationalized at the molecular level why some drugs stimulate the signaling activity of a GPCR (full or partial agonists), whereas others either repress the receptor (inverse agonists) or have no effect on the intrinsic signaling activity (neutral antagonists). Moreover, the existence of a high basal activity of some GPCRs (3) suggests that the conformational transitions leading to activation may occur spontaneously, even in the absence of ligands, which in turn raises questions about the mechanistic roles of GPCR ligands. Understanding the mechanisms and pathways of receptor activation or deactivation, and how these are linked to the binding of ligands with different chemical structures and pharmacological efficacies, will aid in design of new GPCR-targeted drugs with tailored pharmacological responses and fewer side effects. To attain these goals, new methods are required to visualize the conformational dynamics of GPCRs in the presence and absence of drugs.The β 2 -adrenergic receptor (β 2 AR) has been extensively investigated in crystals (4, 5), by NMR in solution (6-11), by bulk fluorescence spectroscopy in s...

[reaction: see text]Reaction of indole amides 5 with tributylstannane gave spiroindolenines 9 which are readily converted into spiropyrrolidinyloxindoles. This tricyclic system is found in a number of interesting natural products.

A new concept for sequence‐specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S‐adenosyl‐L‐methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase‐catalyzed sequence‐specific coupling with DNA. Sequence‐specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.

Sequence-specific labeling of DNA is of immense interest for analytical and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M.TaqI). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5'-TCGA-3' DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1''-(N''-dansyl)-4''-aminobutyl]-5'-(1-aziridinyl)-5'-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M.TaqI and is coupled with a short duplex oligodeoxynucleotide by M.TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M.TaqI-catalyzed reaction.

Movie S1. Addition of antibody EP2-19G2 to a solution of the stilbene hapten 1 under UV illumination illustrates the striking difference between the faint purple fluorescence of 1 in absence of antibody and the intense powder-blue luminescence of the antibody-stilbene complex.

Single-molecule fluorescence resonance energy transfer studies of freely diffusing hairpin ribozymes with different combinations of helical junction and loop elements reveal striking differences in their folding behavior. We examined a series of six different ribozymes consisting of two-, three-and four-way junction variants, as well as corresponding constructs with one of the two loops removed. Our results highlight the varying contributions of preformed secondary structure elements to tertiary folding of the hairpin ribozyme. Of the three helical junction variants studied, the four-way junction strongly favored folding to a docked conformation of the two loops, required for catalytic activity. Moreover, the four-way junction was uniquely able to fold to a similar compact structure even in the absence of specific loop-loop docking interactions. A key feature of the data is the observation of broadening/tailing in the fluorescence resonance energy transfer histogram peak for a single-loop mutant of the four-way junction at higher Mg 21 concentrations, not observed for any of the other single-loop variants. This feature is consistent with interconversion between compact and extended structures, which we estimate takes place on the 100ms timescale using a simple model for the peak shape. This unique ability of the four-way junction ribozyme to populate an undocked conformation with native-like structure (a quasi-docked state) likely contributes to its greater tertiary structure stability, with the quasi-docked state acting as an intermediate and facilitating the subsequent formation of the specific hydrogen bonding network during docking of the two loops. The inability of two-and three-way junction ribozymes to fully populate a docked conformation reveals the importance of correct helical junction geometry as well as loop elements for effective ribozyme folding.

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short ( approximately 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.

RNA conformational transformations are integral to RNA's biological functions. Further, structured RNA molecules exist as a series of dynamic intermediates in the course of folding or complexation with proteins. Thus, an understanding of RNA folding and function will require deep and incisive understanding of its dynamic behavior. However, existing tools to investigate RNA dynamics are limited. Here we introduce a powerful fluorescence polarization anisotropy approach that utilizes a rare base analog that retains substantial fluorescence when incorporated into helices. We show that 6-methylisoxanthopterin (6-MI) can be used to follow the nanosecond dynamics of individual helices. We then use 6-MI to probe the dynamics of an individual helix, referred to as P1, within the 400nt Tetrahymena group I ribozyme. Comparisons of the dynamics of the P1 helix in wild type and mutant ribozymes and in model constructs reveal a highly immobilized docked state of the P1 helix, as expected, and a relatively mobile ‘open complex’ or undocked state. This latter result rules out a model in which slow docking of the P1 helix into its cognate tertiary interactions arises from a stable alternatively docked conformer. The results are consistent with a model in which stacking and tertiary interactions of the A3 tether connecting the P1 helix to the body of the ribozyme limit P1 mobility and slow its docking, and this model is supported by cross-linking results. The ability to isolate the nanosecond motions of individual helices within complex RNAs and RNA/protein complexes will be valuable in distinguishing between functional models and in discerning the fundamental behavior of important biological species.