Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments.
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Bacterial Proteins↗
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FRET probes to monitor phospholipase A2 activity.
Phosphatidylethanolamine and -choline derivatives equipped with fluorescent donor-acceptor pairs of dyes connected to the tips of the fatty acids were synthesised and shown to be suitable substrates for phospholipase A2.
Fluorescence Resonance Energy Transfer↗
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.
Fluorescein↗
Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences.
We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design.
Base Sequence↗
Imaging protein-protein interactions in cell motility using fluorescence resonance energy transfer (FRET).
Protein-protein interactions and signal transduction pathways have traditionally been analysed using biochemical techniques or standard microscopy. Although invaluable in the delineation of protein hierarchy, these methods do not provide information on the true spatial and temporal nature of complex formation within the intact cell. Recent advances in microscopy have allowed the development of new methods to analyse protein-protein interactions at very high resolution in both fixed and live cells. The present paper provides a brief overview of using fluorescence resonance energy transfer to analyse directly molecular interactions and conformational changes in various proteins involved in the regulation of cell adhesion and motility.
Cell Adhesion↗
Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome.
During protein biosynthesis, nascent protein chains are directed along a long narrow tunnel that spans the large ribosomal subunit. It has recently become clear that this structural feature has evolved to effect regulatory control over aspects of protein synthesis and protein trafficking. Since this control is nascent chain-specific, ribosomal components that form the tunnel must be involved in recognizing selected nascent proteins as they pass by. The present study focuses on one such situation in which nascent secretory proteins and membrane proteins are distinguished by the ribosome-induced folding of the latter's hydrophobic transmembrane sequence far inside the ribosomal tunnel and close to the peptidyltransferase centre.
Animals↗
[FRAP, FLIP, FRET, BRET, FLIM, PRIM...new techniques for a colourful life].
Cell and tissue imaging provides scientists with wonderful tools, thanks to a fruitful dialog between chemistry, optical, mechanical, computational sciences and biology. Confocal microscopy, videomicroscopy together with a new generation of fluorochromes (especially those derived from green fluorescent protein, GFP) and image analysis software allow to visualize life in all its dimensions (space and time). Cell imaging also allows to quantify biological processes at the cellular level, to analyse both stoechiometry and dynamics of molecular interactions involved in cell and tissue regulations. Entering the new era of post-genomics requires a better knowledge of advantages and limitations of these new approaches.
Cell Physiological Phenomena↗
The fluorescence resonance energy transfer (FRET) gate: a time-resolved study.
The two-step energy-transfer process in a self-assembled complex comprising a cationic conjugated polymer (CCP) and a dsDNA is investigated by using pump-dump-emission spectroscopy and time-correlated single-photon counting; energy is transferred from the CCP to an ethidium bromide (EB) molecule intercalated into the dsDNA through a fluorescein molecule linked to one terminus of the DNA. Time-dependent anisotropy measurements indicate that the inefficient direct energy transfer from the CCP to the intercalated EB results from the near orthogonality of their transition moments. These measurements also show that the transition moment of the fluorescein spans a range of angular distributions and lies between that of the CCP and EB. Consequently, the fluorescein acts as a fluorescence resonance energy-transfer gate to relay the excitation energy from the CCP to the EB.
DNA↗
Allosteric mechanism in AMPA receptors: a FRET-based investigation of conformational changes.
Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.
Allosteric Regulation↗
Confirmation by FRET in individual living cells of the absence of significant amyloid beta -mediated caspase 8 activation.
When cells are exposed to death-inducing molecules such as tumor necrosis factor-alpha or Fas, caspase 8 is activated and cleaves an apoptotic facilitator, Bid, that is a member of the Bcl-2 family. After additional modification, the C-terminal moiety of Bid is translocated to the mitochondria and induces the release of cytochrome c into the cytoplasm. In an attempt to directly observe the cleavage of Bid and the following events in living cells, we constructed a vector that encoded Bid fused with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) (YFP-Bid-CFP). On expression of YFP-Bid-CFP in mammalian cells, we were able to observe the efficient transfer of energy from excited CFP to YFP within the YFP-Bid-CFP molecule and, importantly, the fusion protein YFP-Bid-CFP was fully functional in cells. When YFP-Bid-CFP was cleaved by caspase 8, on activation by anti-Fas Abs but not by Abeta or tunicamycin, no such transfer of energy was detected. To our knowledge, this is the first report of (i) visualization of the activation of Bid by proteolytic cleavage, with direct observation of the cleavage of YFP-Bid-CFP in the cytoplasm and subsequent translocation of the cleaved Bid to mitochondria and (ii) the absence of Abeta- or tunicamycin-mediated significant activation of caspase 8 in individual living cells.
3T3 Cells↗
FRET analysis of in vivo dimerization by RNA-editing enzymes.
Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo- and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent.
Adenosine Deaminase↗
Using fluorescence resonance energy transfer (FRET) for measuring 2-5A analogues ability to activate RNase L.
The development of a method for measuring the ability of 2-5A analogues to activate the cleavage of an oligoribonucleotide substrate by RNase L is described. This method is based on fluorescence resonance energy transfer. The method is easily performed with 96-well plates, allowing for quantitative high-throughput analyses of 2-5A analogues under different reaction conditions.
Adenine Nucleotides↗
"Fret no more my child ... for I'm all over heaven all day": religious beliefs in the bereavement of African American, middle-aged daughters coping with the death of an elderly mother.
This article examines the ways in which religious beliefs of 30 African American, middle-aged daughters help them cope with the death of their elderly mothers. This qualitative, exploratory study found that daughters use their beliefs to move through states of grief that allow them to prepare, relinquish control, accept death, and maintain a connection to their mothers beyond death. Important themes identified in this study include the belief in an after life and the reunification of family members there. Findings suggest that religious beliefs provide a means for adult daughters to cope with the tasks of living in the present yet maintain a tie with their deceased mothers that serves to enhance their religious beliefs and fortitude in daily living.
Adaptation, Psychological↗
Design of multidye systems for FRET-based applications.
A new solid phase approach, based on orthogonal protective group strategy utilizing Fmoc and DMTr groups, was used to assemble linear polymeric chains with pending groups at desired locations. A compound synthesized using four different fluorophores with consequently overlapping absorption and emission spectra (pyrene, perylene, fluorescein and TAMRA) was shown to fluoresce at 570 nm when excited at 330 nm, demonstrating sequential energy transfer across four chromophores.
Energy Transfer↗
Four color fret dye nucleotide terminators for DNA sequencing.
The synthesis of four color set of energy transfer-dye terminators (8a-8d) starting from p-iodo-beta-phenylalanine was accomplished and their utility in the sequencing reactions has been evaluated.
Energy Transfer↗
Synthesis and energy transfer efficiency of FRET terminators derived from different linkers.
A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5'-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.
Base Sequence↗
Perylene attached to DNA through stiff or flexible linker: duplex stability and FRET.
Two fluorescent nucleosides, 5-(perylen-3-ylethynyl)-2'-deoxyuridine and 5-[(perylen-3-yl)methoxypropyn-1-yl]-2'-deoxyuridine, were incorporated into synthetic oligodeoxyribonucleotides and spectral properties of the conjugates and their duplexes were studied.
DNA↗
Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy.
During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques-two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy-to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag-Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein-protein and -membrane interactions involved in the formation of complex macromolecular structures.
Animals↗