FRET Technique Research Articles

Antagonistic cannabinoid CB 1/dopamine D 2 receptor interactions in striatal CB 1/D 2 heteromers. A combined neurochemical and behavioral analysis

In vitro results show the ability of the CB 1 receptor agonist CP 55,940 to reduce the affinity of D 2 receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D 2 receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB 1–D 2 receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB 1/D 2 receptor–receptor interactions may occur in CB 1/D 2 formed heteromers. Antagonistic CB 1/D 2 interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB 1 receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB 1 receptor antagonist rimonobant but also the specific A 2A receptor antagonist MSX-3 blocked the inhibitory effect of CB 1 receptor agonist on D 2-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB 1/D 2 receptor–receptor interactions within CB 1/D 2 heteromers in which A 2A receptors may also participate.

Fluorescence Resonance Energy Transfer Analysis of Bid Activation in Living Cells during Ultraviolet-induced Apoptosis

Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both the membrane death receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic Bcl-2 family member, is important in most cell types to apoptosis in response to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, comprised of yellow and cyan fluorescent protein and a full length Bid, was used as a fluorescence resonance energy transfer analysis (FRET) probe. Using the FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated at 9+/-1 h after UV irradiation, and the average duration of the activation was 75+/-10 min. Bid activation coincided with a collapse of the mitochondrial membrane potential with an average duration of 50+/-10 min. When cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the process of Bid activation was completely inhibited, but the apoptosis was only partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) did not block Bid activation. Furthermore, the endogenous Bid activation with or without Z-IETD-fmk in response to UV irradiation was confirmed by Western blotting. In summary, using the FRET technique, we observed the dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependent event.

Quantifying the relation between adhesion ligand-receptor bond formation and cell phenotype.

One of the fundamental interactions in cell biology is the binding of cell receptors to adhesion ligands, and many aspects of cell behavior are believed to be regulated by the number of these bonds that form. Unfortunately, a lack of methods to quantify bond formation, especially for cells in 3D cultures or tissues, has precluded direct probing of this assumption. We now demonstrate that a FRET technique can be used to quantify the number of bonds formed between cellular receptors and synthetic adhesion oligopeptides coupled to an artificial extracellular matrix. Similar quantitative relations were found between bond number and the proliferation and differentiation of MC3T3-E1 preosteoblasts and C2C12 myoblasts, although the relation was distinct for each cell type. This approach to understanding 3D cell-extracellular matrix interactions will allow one to both predict cell behavior and to use bond number as a fundamental design criteria for synthetic extracellular matrices.

Chromatin Compaction at the Mononucleosome Level

Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histones. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker histone H1, and histone modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Histone H1 leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histones with histones prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histones have few modifications in the core domain and form nucleosomes normally. Acetylating all histones or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp.

Detecting and Imaging Protein-Protein Interactions During G Protein-Mediated Signal Transduction In Vivo and In Situ by Using Fluorescence-Based Techniques

An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as bimolecular fluorescence complementation (BiFC), can provide information about where and when protein-protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein- mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal transduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies.

Screening of drugs by FRET analysis identifies inhibitors of SARS-CoV 3CL protease

SARS-CoV 3CL protease is essential for viral protein processing and is regarded as a good drug target to prevent SARS-CoV replication. In the present study, we established a high-throughput FRET technique for screening for anti-SARS-CoV 3CL protease drugs. Of a thousand existing drugs examined, hexachlorophene was identified as the most potent in inhibiting SARS-CoV 3CL protease. Further characterization showed that it was effective at micromolar concentrations ( K i = 4 μM). The binding mode was competitive, and the inhibitory effect was dependent on preincubation time. Two other drugs, triclosan and nelfinavir, were about 10 times less potent. The structure-based search and biological evaluation of various hexachlorophene analogues were described. These analogues gave optimal inhibitory activity against SARS-CoV 3CL protease with IC 50 values ranging from 7.6 to 84.5 μM. Optimization of hexachlorophene analogues was shown to provide several active 3CL protease inhibitors that function as potential anti-SARS agents.

FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1

Lipoproteins or lipopeptides (LP) are bacterial cell wall components detected by the innate immune system. For LP, it has been shown that TLR2 is the essential receptor in cellular activation. However, molecular mechanisms of LP recognition are not yet clear. We used a FLAG-labeled derivative of the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (Pam(3)CSK(4)) to study the roles of CD14, TLR2 and TLR1 in binding and signaling of LP and their molecular interactions in human cells. The activity of Pam(3)CSK(4)-FLAG was TLR2 dependent, whereas the binding was enabled by CD14, as evaluated by flow cytometry and confocal microscopy. Using FRET and FRAP imaging techniques to study molecular associations, we could show that after Pam(3)CSK(4)-FLAG binding, CD14 and Pam(3)CSK(4)-FLAG associate with TLR2 and TLR1, and TLR2 is targeted to a low-mobility complex. Thus, LP binding to CD14 is the first step in the LP recognition, inducing physical proximity of CD14 and LP with TLR2/TLR1 and formation of the TLR2 signaling complex.

Parallel vs. anti-parallel orientation in a curdlan/oligo(dA) complex as estimated by a FRET technique

We already found that beta-1,3-glucan polysaccharides form polymeric complexes with certain polynucleotides, but the parallel vs. anti-parallel orientation in those complexes had remained unsolved. In this paper, this controversial problem has been discussed for curdlan/oligo(dA) complexes utilizing two different energy transfer techniques. The first system consists of a combination of fluorescein-labeled curdlan and 3'-(or 5'-)tetramethyl-rhodamine (TAMRA)-labeled oligo(dA). The second system utilizes gold nanoparticles: that is, two curdlan chains were linked by a disulfide bond and after complexation with oligo(dA), the complex was immobilized on gold nanoparticles. In this system, TAMRA was attached to the 3'(or 5') end of oligo(dA) and the gold particle acted as a fluorescence quencher (energy acceptor). These experiments have led us to conclude that in the curdlan/oligo(dA) complex, parallel orientation is more favourable than anti-parallel orientation. These findings have enabled us to envision a clearer image for the complexation mode between beta-1,3-glucan polysaccharides and polynucleotides.

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

682. New Histidylated Cationic Lipids for DNA-and mRNA- Based Lipofection

Plasmid delivery into the cytosol remains one of the limiting factor to achieve efficient transfection. We have previously demonstrated that the presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers significantly enhances their gene delivery efficiencies. We designed and synthesized two novel non-glycerol and a cholesterol based histidylated cationic amphiphiles containing a single histidine head group. Physico-chemical characteristics of all the novel liposomes and lipoplexes including lipid:DNA interactions, global surface charge, sizes, etc. were evaluated. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-Histidine(N,N-di-n-hexadecylamine, N-methyl)ethylamide (lipid 2) in combination with cholesterol and Cholesteryl-L-Histidine-Ethylamide (lipid 3) in combinaison with DOPE gave efficient DNA and mRNA transfections into various cell lines. DNA transfection efficiency into A549, 293T7 and HeLa cells of Chol/lipid 1 lipoplexes was similar with that of FuGENE6 and DC-Chol lipoplexes but was two order of magnitude higher in HepG2 cells. Membrane fusion activity measurements using FRET technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5–7. By using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter, we showed that the release of DNA from the endosomally trapped lipoplexes to the cytosol is acidic dependent and presumably mediated by the imidazole ring protonation of histidine head group of these cationic amphiphiles. A better efficiency was obtained with Chol/lipid 2 lipoplexes than with Chol/lipid 1 lipoplexes when using the cytosolic luciferase expression vector. As anticipated, transfection efficiency of Lipid 3 was greatly inhibited in the presence of Bafilomycin A1. By contrast, endosome escape of DNA with a new cholesterol based cationic lipid containing no histidine head-group (Alanine-Cholesteryl-Ethylamide; lipid 4) seemed to be independent of endosome acidification. However, transfection efficacy of lipids 3 & 4 was similar. In conclusion, we show that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds or cholesterol is sufficient to impart remarkable transfection properties on the resulting cationic amphiphile via endosome-disrupting characteristics of the histidine functionalities Figure 1

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.

1SB05 一分子FRET法を用いた運動中のキネシンの構造変化の観察(分子モーター研究の新潮流)

1SB05 一分子FRET法を用いた運動中のキネシンの構造変化の観察(分子モーター研究の新潮流)

3P176 歩行中のキネシン分子内部での構造変化の一分子観察(分子モーター)

3P176 歩行中のキネシン分子内部での構造変化の一分子観察(分子モーター)

DNA Binding of a Non-sequence-specific HMG-D Protein is Entropy Driven with a Substantial Non-electrostatic Contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 °C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity

This overview unit focuses on the basics of fluorescence and of the FRET phenomenon, and on methods for detecting FRET and data interpretation. FRET is very versatile and there are more application of these basics than can be covered in a single overview. However, some examples are given of applications of various FRET techniques.

FRETによるGroEL内部での基質タンパク質の運動解析

FRETによるGroEL内部での基質タンパク質の運動解析

Kappa-squared: from nuisance to new sense

The orientation factor, which is commonly called kappa-squared, is often considered to be a nuisance because it represents a significant uncertainty in the distance obtained with the FRET technique. It is shown that this uncertainty is rather small in almost all cases of practical interest if one takes the width of a 67% confidence interval (CI) for the distance distribution as a measure of uncertainty. Kappa-squared has the potential to open up new information on orientations and rotations from time-resolved studies of donor and acceptor anisotropies. One can make sense of such data by designing matrix models for the transitions between states describing various orientations and positions of donors and acceptors in the system.

Correlation of GM-CSF mRNA in bronchoalveolar fluid with indices of clinical activity in sarcoidosis.

Granulocyte-macrophage colony stimulating factor (GM-CSF) has several proinflammatory effects, some of which potentially favour granuloma formation. Its mRNA expression by the inflammatory cells recovered from lungs of patients with pulmonary sarcoidosis has been previously reported. In this study an association between GM-CSF expression and manifestations of the disease was explored. GM-CSF mRNA was detected by reverse transcription polymerase chain reaction in the cells of bronchoalveolar lavage (BAL) fluid of 20 patients with pulmonary sarcoidosis. GM-CSF mRNA expression was positive in 15 of 20 patients with sarcoidosis. Fourteen of the 15 patients with positive mRNA expression had worsening or unchanged disease during the year preceding this study, on the basis of radiographic or physical findings, or both, whereas all five "negative" patients were judged to be improving. Similarly, serum levels of angiotensin converting enzyme, the proportion of lymphocytes in BAL fluid, and the CD4+/CD8+ ratio of lymphocytes in BAL fluid were significantly higher in the positive patients. There was an association between the presence of GM-CSF mRNA in the cells in BAL fluid and other indices of disease activity in sarcoidosis.