FRET Methods Research Articles

1SQ-01 Folding and Dynamics of Globular Proteins Studied by time resolved FRET Methods(1SQ Israel-Japan Joint Symposium on Biophysics "Protein Dynamics: From single molecules to whole cell",The 49th Annual Meeting of the Biophysical Society of Japan)

1SQ-01 Folding and Dynamics of Globular Proteins Studied by time resolved FRET Methods(1SQ Israel-Japan Joint Symposium on Biophysics "Protein Dynamics: From single molecules to whole cell",The 49th Annual Meeting of the Biophysical Society of Japan)

Resting State Orai1 Diffuses as Homotetramer in the Plasma Membrane of Live Mammalian Cells

Store-operated calcium entry is essential for many signaling processes in nonexcitable cells. The best studied store-operated calcium current is the calcium release-activated calcium (CRAC) current in T-cells and mast cells, with Orai1 representing the essential pore forming subunit. Although it is known that functional CRAC channels in store-depleted cells are composed of four Orai1 subunits, the stoichiometric composition in quiescent cells is still discussed controversially: both a tetrameric and a dimeric stoichiometry of resting state Orai1 have been reported. We obtained here robust and similar FRET values on labeled tandem repeat constructs of Orai1 before and after store depletion, suggesting an unchanged tetrameric stoichiometry. Moreover, we directly visualized the stoichiometry of mobile Orai1 channels in live cells using a new single molecule recording modality that combines single molecule tracking and brightness analysis. By alternating imaging and photobleaching pulses, we recorded trajectories of single, fluorescently labeled Orai1 channels, with each trajectory consisting of bright and dim segments, corresponding to higher and lower numbers of colocalized active GFP label. The according brightness values were used for global fitting and statistical analysis, yielding a tetrameric subunit composition of mobile Orai1 channels in resting cells.

Single-Molecule Study of Viomycin’s Inhibition Mechanism on Ribosome Translocation

Viomycin belongs to the tuberactinomycin family of antibiotics against tuberculosis. However, its inhibition mechanism remains elusive. Although it is clear that viomycin inhibits the ribosome intersubunit ratcheting, there are contradictory reports about whether the antibiotic viomycin stabilizes the tRNA hybrid or classical state. By using a single-molecule FRET method to directly observe the tRNA dynamics relative to ribosomal protein L27, we have found that viomycin trapped the hybrid state within certain ribosome subgroups but did not significantly suppress the tRNA dynamics. The persistent fluctuation of tRNA implied that tRNA motions were decoupled from the ribosome intersubunit ratcheting. Viomycin also promoted peptidyl-tRNA fluctuation in the posttranslocation complex, implying that, in addition to acylated P-site tRNA, the decoding center also played an important role of ribosome locking after translocation. Therefore, viomycin inhibits translocation by trapping the hybrid state in the pretranslocation complex and disturbing the stability of posttranslocation complex. Our results imply that ribosome translocation is possibly a synergistic process of multiple decoupled local dynamics.

Bio‐Orthogonal Protein Labeling Methods for Single Molecule FRET

AbstractSingle‐molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique for visualizing the dynamics and architecture of proteins, multi‐subunit protein complexes, or protein‐nucleic acid complexes. To suffice a FRET study, selective labeling with a pair of a donor dye and an acceptor dye is required, with the two dyes on two sites on a protein, or with the donor on one protein sub‐unit while the acceptor on another subunit, or with the pair divided between protein and DNA (RNA). Due to the availability of orthogonal chemical moieties, most biophysicists armed with single FRET have been limited to the processes of nucleic acids metabolism by studying protein‐DNA (‐RNA) interactions. Nevertheless, diverse biological processes usually involve communications between proteins. Indeed, protein‐protein interactions constitute fundamental questions regarding intra‐ and intercellular signal transduction and are under intensive research, yet mainly by conventional methodologies. In this review, various bio‐orthogonal methods recently developed for labeling proteins via protein domains, small peptides, or single amino acids are examined. These approaches comprise a repertoire that overcomes the challenges of labeling two sites on a protein or a protein complex to facilitate the investigation of protein‐protein interactions by the modern single molecule FRET method.

Electrochemistry and in situ fluorescence microscopy of octadecanol layers doped with a BODIPY-labeled phospholipid: Investigating an adsorbed heterogeneous layer

A heterogeneous floating multilayer created from a 99:1 mole mixture of 1-octadecanol and BODIPY-HPC was characterized by fluorescence microscopy. The monomer and the D II dimer emission of the BODIPY moiety displayed a variety of structures including phase separated fluid-like regions (dimer-rich) and more rigid solid-like regions (monomer-rich). These layers were deposited onto a Au(1 1 1) electrode surface and characterized using capacitance measurements and in situ fluorescence microscopy. A direct comparison between the floating layer and the resulting deposited layer revealed an expansion of 1.3 – 2 × with distortion in the fluorescent features due to the multilayer nature of the floating layer. A similar variety of structures or phases were observed to exist on the electrode which displayed different potential dependent changes in fluorescence. The monomer and dimer fluorescence intensity and their ratio were used to characterize the potential dependence of the various adsorbed phases. An example is presented that illustrates the influence of the solid substrate on the characteristics of the adsorbed layer. A region in the floating layer that appeared to be mixed in composition was transformed into a dimer-rich region that had potential dependent characteristics of both. This method is useful for probing the heterogeneity of organic layers adsorbed on electrode surfaces. The phase characteristics of the adsorbed organic layer can be probed using these types of dimer forming fluorescent probes and FRET methods but are complicated by the influence of the solid substrate and the photobleaching that occurs.

Measuring the Energetics of Membrane Protein Dimerization in Mammalian Membranes

Thus far, quantitative studies of lateral protein interactions in membranes have been restricted peptides or simplified protein constructs in lipid vesicles or bacterial membranes. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize the quantitative imaging FRET (QI-FRET) method. The experiments are described in a step-by-step protocol. The protein characterized is the transmembrane domain of glycophorin A, the most extensively studied membrane protein, known to form homodimers in hydrophobic environments. The results suggest that molecular crowding in cellular membranes has a dramatic effect on the strength of membrane protein interactions.

Single-molecule, real-time measurement of enzyme kinetics by alternating-laser excitation fluorescence resonance energy transfer

Using a single-molecule fluorescence method uniquely suitable for binding assay, alternating-laser excitation fluorescence resonance energy transfer (ALEX-FRET), we accurately measured the cleavage rate of 8-17 deoxyribozyme, an RNA-cleaving enzyme, at the single-molecule level in real time with a minimum consumption of samples, i.e., at least three orders of magnitude smaller than used in the conventional ensemble FRET method.

The Role of Bulges and Hinges in RNA Folding

Long-range tertiary interactions govern RNA structural assembly, which is a critical step toward RNA biological functionality. Thereby, a universal strategy has emerged for global conformational change; flexible junctions enable unpaired nucleotides to act as beacons between helical regions. Bulges, for example, are versatile secondary structural elements implicated in helix recognition and packing. The P4-P6 domain of the Tetrahymena ribozyme utilizes this folding strategy by hinging to form two inter-helical tertiary contacts, the adenine-rich (A-rich) bulge and the tetraloop-tetraloop receptor interactions. To explore the kinetic and thermodynamic properties of tertiary contact formation, we probe the P4-P6 domain hinging and ribose zippering that forms the A-rich bulge interaction using single-molecule FRET methods. We obtain the docking and undocking rates of the A-rich bulge and P4 helix as function of cation concentration and temperature. Docking is accelerated and undocking decelerated by Mg2+. In spite of rapid docking at high [Mg2+] (kdock = 20 ± 2 s−1), the A-rich bulge interaction is only marginally stable (Kdock = 1.2 ± 0.1). These results support that the role of the A-rich bulge is to kinetically direct P4-P6 domain folding while thermodynamic stability is added through the tetraloop-receptor interaction. Formation of the A-rich bulge contact shows specificity for divalent cations, with a preference for Mg2+ as anticipated from the Mg2+ coordination observed in structural data. A significant kinetic heterogeneity is characterized; only 50% of the molecules exhibit efficient folding at high [Mg2+]. Mutations of the A-rich bulge construct reveal a crucial role of the P4-P6 secondary architecture in enabling the A-rich bulge contact.

Single Molecule FRET Studies of RNA Tertiary Folding: Elucidating the Thermodynamic and Kinetic Role of Na+ Cations

Understanding the kinetics and thermodynamics of the stabilizing interactions in non-coding RNA provides crucial information about their structural dynamics and ultimately, their biological function. The use of single-molecule FRET methods allows us to investigate the real-time folding and unfolding of an isolated GAAA tetraloop-receptor interaction in the Tetrahymena ribozyme. Cation-dependent folding studies of this ubiquitous interaction show that increasing concentrations of Na+ significantly increase the rate constant for docking and slightly decrease the rate constant for undocking. We examine the temperature-dependence of this Na+-induced folding in order to determine the thermodynamic parameters associated with the folding and unfolding processes. At 150 mM Na+ the folding process is exothermic (ΔH° = −20 kcal/mol) but with a significant entropic cost (ΔS° = −67 cal/mol K), leading to a near zero ΔG° at 298 K. Increasing concentrations of Na+ dramatically increase both ΔH° and ΔS°, with the competition yielding a decrease in ΔG°. For example, by 600 mM Na+ the folding process even becomes endothermic (ΔH° = 4.3 kcal/mol) and entropically rewarding (ΔS° = 20 cal/mol K), with the folding process now becoming slightly favorable (ΔG° = −1.7 kcal/mol). These results indicate that increasing Na+ concentration favors folding by increasing entropic gains more than enthalpic losses, which leads to a more favorable folding free energy change. We propose a model that uses the competing roles of solvent and structure to explain these gains and losses.

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase

The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stopped-flow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8- to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/RNAP-promoter rotational changes during 5- to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.

Time-resolved FRET method for typing polymorphic alleles of the human leukocyteantigen system by using a single DNAprobe

By tens-of-picosecond resolved fluorescence detection we study Förster resonance energy transfer between a donor and a black-hole-quencher acceptor bound at the 5'- and 3'-positions of a probe-oligonucleotide matching an allelic region of a class II antigen of the human leukocyte antigen system, namely the region between codons 52 and 57 of the HLA DQB1 0201 allele. This dual-labelled probe is annealed with either the exactly complementary allelic sequence or with polymorphic sequences of the same region. We detect the longest-lived donor fluorescence in the case of the perfect hybridization achieved with the 0201 allele and definitely faster and distinct decays for the other allelic variants, some of which are single-nucleotide polymorphic variants.

Two‐sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, beta1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of beta1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of beta1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-by-pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between beta1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between beta1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of beta1-integrin-ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with beta1-integrins.

Fluorescence-based melting assays for studying quadruplex ligands

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for T m calculation, complicating a direct comparison of the results obtained by different laboratories.

Carboxyl-modified single-walled carbon nanotubes selectively induce human telomeric i-motif formation.

As the leading nanodevice candidate, single-walled carbon nanotubes (SWNTs) have potential therapeutic applications in gene therapy and novel drug delivery. We found that SWNTs can inhibit DNA duplex association and selectively induce human telomeric i-motif DNA formation by binding to the 5'-end major groove under physiological conditions or even at pH 8.0. SWNT binding to telomeric DNA was studied by UV melting, NMR, S1 nuclease cleavage, CD, and competitive FRET methods. These results suggest that SWNTs might have the intriguing potential to modulate human telomeric DNA structures in vivo, like biologically relevant B-A and B-Z DNA transitions, which is of great interest for drug design and cancer therapy.

Nanoparticle based detection of multiprotein complexes on DNA

Protein-DNA interactions as well as protein-protein interactions play critical roles in biological processes and have been extensively studied using solution-based assays. These assays, however, often lack the ability to be introduced in vivo due to the lack of proper experimental methods, and are limited both in the distances which can be probed and the number of partners which can be investigated. We are developing novel approaches to studying protein-DNA interactions involving multi-protein complexes, with applications to cell biology and drug discovery. Our approach relies on the energy transfer between single metallic nanoparticles positioned onto DNA, site specifically and terminally, and dye-labeled proteins. This energy transfer process is efficient over longer distances than classical FRET methods enhancing our ability to monitor protein-DNA interactions and is readily applicable to multiplexing (probing multiple proteins binding to the same DNA molecule). Our initial experiments have involved well-known DNA binding proteins such as restriction endonucleases and zinc fingers. Based on previous work demonstrating the cellular acceptance of both fluorescent dyes and nanoparticles, we are developing this technology to probe protein/DNA interactions in the cell and for drug screening applications.

Rhodopsin self-associates in asolectin liposomes.

We show that the photoreceptor rhodopsin (Rh) can exist in the membrane as a dimer or multimer using luminescence resonance energy transfer and FRET methods. Our approach looked for interactions between Rh molecules reconstituted into asolectin liposomes. The low receptor density used in the measurements ensured minimal receptor crowding and artifactual association. The fluorescently labeled Rh molecules were fully functional, as measured by their ability to activate the G protein transducin. The luminescence resonance energy transfer measurements revealed a distance of 47-50 Angstroms between Rh molecules. The measured efficiency of FRET between receptors was close to the theoretical maximum possible, indicating nearly quantitative Rh-Rh association. Together, these results provide compelling evidence that Rh spontaneously self-associates in membranes.

Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET

Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg2+, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, conformational heterogeneity similar to that noted in more complex RNA systems is observed; relatively rapid docking/undocking transitions are detected for approximately two-thirds of the RNA molecules, with significant subpopulations exhibiting few or no transitions on the 10- to 30-s time scale for photobleaching. The rate constants are determined from analysis of probability densities, which allows a much wider range of time scales to be analyzed than standard histogram procedures. The data for the GAAA tetraloop receptor are compared with kinetic and equilibrium data for other RNA tertiary interactions.

FRET measurements of trapped oligonucleotide duplexes

Sensitive methods developed to measure laser-induced fluorescence from trapped ions have been applied to study the intermediate states of the dissociation of oligonucleotide duplexes. Double-stranded oligonucleotide anions were prepared with FRET donor/acceptor pairs attached. Gas phase ions were generated by electrospray ionization and injected into a heated quadrupole ion trap where they were stored and exposed to Nd:YAG laser pulses at 532 nm. The dissociation of the duplexes into single strands was monitored by both fluorescence and mass spectra. The comparison of the degree of dissociation of the duplex observed in the mass spectra to the fluorescence intensity of the donor allows for the determination of the intermediate conformations of the unzipping duplex. The observation of changes in the donor fluorescence in the absence of single strands correlates with an intermediate state characterized by threshold unzipping at the weaker binding end of the duplex. These data are consistent with a model of intermediate state formation as a function of temperature. These studies suggest that FRET methods will be useful to obtain direct measurements, which characterize the dynamics of conformational changes of biomolecules in the gas phase.

Retinoid X Receptor (RXR) Agonist-induced Antagonism of Farnesoid X Receptor (FXR) Activity due to Absence of Coactivator Recruitment and Decreased DNA Binding

The bile salt export pump (BSEP) plays an integral role in lipid homeostasis by regulating the canalicular excretion of bile acids. Induction of BSEP gene expression is mediated by the farnesoid X receptor (FXR), which binds as a heterodimer with the retinoid X receptor (RXR) to the FXR response element (FXRE) located upstream of the BSEP gene. RXR ligands mimic several partner ligands and show additive effects upon coadministration. Using real-time quantitative PCR and cotransfection reporter assays, we demonstrate that the RXR agonist LG100268 antagonizes induction of BSEP expression mediated by endogenous and synthetic FXR ligands, CDCA and GW4064, respectively. Moreover, this antagonism is a general feature of RXR agonists and is attributed to a decrease in binding of FXR/RXR heterodimers to the BSEP-FXRE coupled with the inability of RXR agonists to recruit coactivators to FXR/RXR. Our data suggest that FXR/RXR is a conditionally permissive heterodimer and is the first example of RXR ligand-mediated antagonism of FXR activity. Because FXR agonists lower triglyceride levels, our results suggest a novel role for RXR-mediated antagonism of FXR activity in the development of hypertriglyceridemia observed with RXR agonists in rodents and humans.