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At least 469 records · Page 26Linked to original sources
PubMed2002

In situ analysis of spatial relationships between proteins of the nuclear pore complex.

Macromolecular transport between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs). The NPC in the budding yeast Saccharomyces cerevisiae is a 60-MDa structure embedded in the nuclear envelope and composed of ~30 proteins, termed nucleoporins or nups. Here we present a large-scale analysis of spatial relationships between nucleoporins using fluorescence resonance energy transfer (FRET) in living yeast cells. Energy transfer was measured in a panel of strains, each of which coexpresses the enhanced cyan and yellow fluorescent proteins as fusions to distinct nucleoporins. With this approach, we have determined 13 nucleoporin pairs yielding FRET signals. Independent experiments are consistent with the FRET results: Nup120 localization is perturbed in the nic96-1 mutant, as is Nup82 localization in the nup116Delta mutant. To better understand the spatial relationship represented by an in vivo FRET signal, we have investigated the requirements of these signals. We demonstrate that in one case FRET signal is lost upon insertion of a short spacer between the nucleoporin and its enhanced yellow fluorescent protein label. We also show that the Nup120 FRET signals depend on whether the fluorescent moiety is fused to the N- or C-terminus of Nup120. Combined with existing data on NPC structure, the FRET pairs identified in this study allow us to propose a refined molecular model of the NPC. We suggest that the approach may serve as a prototype for the in situ study of other large macromolecular complexes.

Bacterial Proteins↗
PubMed1998

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.

Animals↗
PubMed1999

Homogeneous genotyping assays for single nucleotide polymorphisms with fluorescence resonance energy transfer detection.

A homogeneous detection mechanism based on fluorescence resonance energy transfer (FRET) has been developed for two DNA diagnostic tests. In the template-directed dye-terminator incorporation (TDI) assay, a donor dye-labeled primer is extended by DNA polymerase using allele-specific, acceptor dye-labeled ddNTPs. In the dye-labeled oligonucleotide ligation (DOL) assay, a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase. Once the donor and acceptor dyes become part of a new molecule, intramolecular FRET is observed over background intermolecular FRET. The rise in FRET, therefore, can be used as an index for allele-specific ddNTP incorporation or probe ligation. Real time monitoring of FRET greatly increases the sensitivity and reliability of these assays. Change in FRET can also be measured by end-point reading when appropriate controls are included in the experiment. FRET detection proves to be a robust method in homogeneous DNA diagnostic assays.

Animals↗
PubMed2007

Luminescent quantum dots fluorescence resonance energy transfer-based probes for enzymatic activity and enzyme inhibitors.

The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 microg/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.

Cadmium Compounds↗
PubMed1994

The histrionicotoxin-sensitive ethidium binding site is located outside of the transmembrane domain of the nicotinic acetylcholine receptor: a fluorescence study.

A novel, relatively photostable, long-wavelength fluorescent membrane probe, N-(Texas Red sulfonyl)-5(and 6)-dodecanoylamine (C12-Texas Red), was synthesized and used as an electronic energy acceptor for Förster fluorescence resonance energy transfer (FRET) between ethidium bound to a histrionicotoxin-sensitive binding site on the Torpedo nicotinic acetylcholine receptor (AChR) and the lipid membrane surface. FRET from membrane-partitioned 5-(N-dodecanoylamino)fluorescein (C12-fluorescein) to the membrane-partitioned C12-Texas Red was also determined with a parallel set of cuvettes to (1) compare FRET results with a donor in a known position in the membrane and (2) assess the surface density of the membrane-partitioned C12-Texas Red. Stern-Volmer analysis of the FRET results showed that C12-Texas Red quenched membrane-partitioned C12-fluorescein fluorescence 2.9 times more effectively than it quenched the receptor-bound ethidium fluorescence even though the Förster critical distances for the two donor-acceptor pairs were very similar (49.9 and 54.3 A, respectively). Analysis of the ethidium to C12-Texas Red FRET as a function of acceptor surface density with the assumptions that the donor is attached along the major axis of symmetry of a cylindrical protein embedded perpendicularly into the membrane (On-Axis FRET model) suggested that the distance of closest approach between the receptor-bound ethidium and the membrane surface was approximately 52 A. Because the minimum distance between the surface of the lipid-membrane domain and the major symmetry axis of the AChR is approximately 28 A, the FRET results strongly suggest that the ethidium binding site is not located near the entrance of the luminal transmembrane domain is generally assumed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphibian Venoms↗
PubMed2003

Receptor-regulated dynamic interaction between endothelial nitric oxide synthase and calmodulin revealed by fluorescence resonance energy transfer in living cells.

The endothelial isoform of nitric oxide synthase (eNOS), a key regulator of vascular tone, is activated in endothelial cells by diverse Ca(2+)-mobilizing agonists, including vascular endothelial growth factor (VEGF). Although the activation state of eNOS and the subcellular localization of the enzyme are both highly regulated, the relationship between enzyme activity and subcellular targeting remains obscure. We aim here to elucidate this relationship by direct dynamic imaging analysis of Ca(2+)/CaM-dependent eNOS activation in living endothelial cells, using high-resolution confocal microscopy and donor dequenching fluorescence resonance energy transfer (FRET) techniques. Confocal images show a complex pattern of eNOS subcellular distribution; the enzyme is concentrated in both the plasma membrane and internal membranes, with robust expression in the perinuclear region. We construct a fusion protein between eNOS and the FRET-based calcium sensor cameleon, and analyze the temporal and spatial pattern of VEGF-mediated calcium mobilization using donor dequenching FRET methods. We find that VEGF promotes rapid mobilization of intracellular calcium throughout the regions of the cell in which eNOS is distributed. We further create a series of fusion proteins and use FRET imaging methods to study the interactions between eNOS and its obligate allosteric activator protein calmodulin. We clone the FRET acceptor EYFP (enhanced yellow fluorescent protein) at the C-terminus of calmodulin, and the FRET donor ECFP (enhanced cyan fluorescent protein) into eNOS at a site adjacent to its calmodulin-binding domain. FRET imaging analysis of individual endothelial cells cotransfected with eNOS-ECFP and calmodulin-EYFP shows that VEGF induces interactions between eNOS and calmodulin wherever both are present in the cell. Our studies provide evidence that the pool of rapidly responsive receptor-activated eNOS is distributed throughout endothelial cells in both plasma membrane and internal membrane structures, and that this distribution parallels the localization of agonist-induced intracellular Ca(2+) changes in the vicinity of eNOS.

Animals↗
PubMed2003

ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer.

ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of membrane excitability in many cell types. ATP inhibits channel activity by binding to a specific site formed by the N and C termini of the pore-forming subunit, Kir6.2, but the structural changes associated with this interaction remain unclear. Here, we use fluorescence resonance energy transfer (FRET) to study the ATP-dependent interaction between the N and C termini of Kir6.2 using a construct bearing fused cyan and yellow fluorescent proteins (ECFP-Kir6.2-EYFP). When expressed in human embryonic kidney cells, ECFP-Kir6.2-EYFP/SUR1 channels displayed FRET that was augmented by agonist stimulation and diminished by metabolic poisoning. Addition of ATP to permeabilized cells or isolated plasma membrane sheets increased FRET. FRET changes were abolished by Kir6.2 mutations that altered ATP-dependent channel closure and channel gating. In the wild-type channel, the ATP concentrations, which increased FRET (EC(50) = 1.36 mM), were significantly higher than those causing channel inhibition (IC(50) = 0.29 mM). Demonstrating the existence of intermolecular interactions, a dimeric construct comprising two molecules of Kir6.2 linked head-to-tail (ECFP-Kir6.2-Kir6.2-EYFP) displayed less FRET than the monomer in the absence of nucleotide but still exhibited ATP-dependent FRET increases (EC(50) = 1.52 mM) and channel inhibition. We conclude that binding of ATP to Kir6.2, (i). alters the interaction between the N- and C-terminal domains, (ii). probably involves both intrasubunit and intersubunit interactions, (iii). reflects ligand binding not channel gating, and (iv). occurs in intact cells when subplasmalemmal [ATP] changes in the millimolar range.

Adenosine Diphosphate↗
PubMed2004

Apolipoprotein E4 domain interaction occurs in living neuronal cells as determined by fluorescence resonance energy transfer.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer disease. Although the mechanisms remain to be determined, the detrimental effects of apoE4 in neurobiology must be based on its unique structural and biophysical properties. One such property is domain interaction mediated by a salt bridge between Arg-61 in the N-terminal domain and Glu-255 in the C-terminal domain of apoE4. This interaction, which does not occur in apoE3 or apoE2, causes apoE4 to bind preferentially to certain lipoprotein particles in vitro and in vivo. Here we used fluorescence resonance energy transfer (FRET) to determine whether apoE4 domain interaction occurs in living neuronal cells. Neuro-2a cells were transfected with constructs encoding apoE3 or apoE4 in which yellow fluorescent protein (YFP) was fused to the N terminus, and cyan fluorescent protein (CFP) was fused to the C terminus. To generate a FRET signal that can be detected by spectrum confocal microscopy, the labeled N and C termini must be in close proximity (<100 A). FRET signals occurred in cells transfected with YFP-apoE4-CFP but not in those transfected with YFP-apoE3-CFP, suggesting that the N and C termini of apoE4 are in close proximity in living cells and that those of apoE3 are not. FRET signals did not occur in cells cotransfected with YFP-apoE4 and apoE4-CFP, suggesting that the FRET in YFP-apoE4-CFP-transfected cells was intramolecular. Mutation of Arg-61 to Thr or Glu-255 to Ala in apoE4, which disrupts domain interaction, abolished FRET in Neuro-2a cells, strongly suggesting that the FRET in YFP-apoE4-CFP cells was caused by domain interaction. ApoE4-producing cells secreted less phospholipid than apoE3-producing cells, but after disruption of domain interaction in apoE4, phospholipid secretion increased to the levels seen with apoE3, suggesting that domain interaction decreases the phospholipid-binding capacity of apoE4. Thus, apoE4 domain interaction occurs in living neuronal cells and may be a molecular basis for apoE4-related neurodegeneration.

Animals↗
PubMedDate not supplied

Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy.

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

Algorithms↗
PubMed2006

Spectroscopy-based quantitative fluorescence resonance energy transfer analysis.

The combination of green fluorescent protein mutants and fluorescence resonance energy transfer (FRET) forms a powerful tool for ion channel studies. A key to successful application of green fluorescent protein-based FRET is to reliably separate the FRET signal from various non-FRET fluorescence emissions that coexist in any experimental system. This chapter introduces a FRET quantification method that is based on fluorescence spectroscopic microscopy. Application of this "spectra FRET" method to both the confocal imaging of Xenopus oocytes and the epifluorescence imaging of culture cells is described. The fluorescence intensity ratio measurement, a complementary non-FRET method for identifying the channel subunit stoichiometry, is also discussed.

Animals↗
PubMed2006

Fluorescence resonance energy transfer analysis of mitochondrial:lipid association in the porcine oocyte.

The role of endogenous lipid in the provision of energy during in vitro maturation of immature porcine oocytes has been studied. Fluorescence resonance energy transfer (FRET) acceptor bleaching methods have been used to examine mitochondrial:lipid droplet co-localisation in live oocytes. FRET experiments demonstrate whether organelles are within the FRET-distance (i.e. 6-10 nm), thus showing true association on a molecular scale. Immature and in vitro-matured porcine oocytes were stained with Mitotracker Green (MTG; mitochondria) and Nile Red (NR; lipid droplets). The data indicated sufficient overlap between MTG emission and NR excitation to support a FRET reaction and that mitochondria and lipid droplets were sufficiently co-localised for a FRET reaction to occur. When NR-stained lipid droplets were specifically bleached, a significant increase in the MTG signal in stained mitochondria was observed (FRET efficiency, E=22.2 +/- 3.18%). These results strongly suggest a metabolic role for lipid metabolism during oocyte maturation. This conclusion was reinforced by the use of inhibitors of fatty acid beta-oxidation, methyl palmoxirate or mercaptoacetate, exposure to which during oocyte maturation led to developmental failure post-fertilisation. These data provide strong evidence that MTG and NR can act as a FRET pair and that in porcine oocytes, mitochondria and lipid droplets lie within 6-10 nm of each other, indicating association on a molecular scale. The findings also suggest that endogenous triglycerides play an important role in energy metabolism during porcine in vitro maturation.

Aldehydes↗
PubMedDate not supplied

Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.

Bacterial Proteins↗
PubMed2002

Surface fluorescence resonance energy transfer studies on interfacial adsorption of Thermomyces (humicola) lanuginosa lipase, using monomolecular films of cis-parinaric acid.

The fluorescence resonance energy transfer (FRET) technique was adapted to study the process whereby lipase is adsorbed to monomolecular lipid films spread at the air-water interface. When cis-parinaric acid (cis-PnA) was spread over an aqueous subphase before the injection of sodium taurodeoxycholate (NaTDC) and Thermomyces lanuginosa lipase (TLL), no FRET was observed. Under these conditions, no adsorption of TLL was detected using an ELISA. In contrast, FRET occurred when cis-PnA was spread over an aqueous subphase containing NaTDC and TLL. The FRET signals observed were attributed to the interactions between the adsorbed TLL and the cis-PnA monomolecular films. Comparisons between the fluorescence emission spectra corresponding to the bulk phase and the aspirated film, in the presence and absence of TLL, showed that cis-PnA was undetectable in the bulk phase. We concluded that the FRET originated from the interface and not from the bulk phase. Using surface FRET, we estimated that the surface excess of the catalytically inactive mutant, TLL(S146A), was 1.6 higher than that present in the wild-type TLL. This finding is in agreement with independent measurements of the surface excess of TLL and TLL(S146A) on monomolecular films of cis-PnA.

Adsorption↗
PubMed2000

Europium cryptate-tethered ribonucleotide for the labeling of RNA and its detection by time-resolved amplification of cryptate emission.

TRACE (time-resolved amplification of cryptate emission), also called HTRF for pharmaceutical applications, is a homogeneous time-resolved fluorescence technique well adapted for the study of molecular interactions. It is based on fluorescence resonance energy transfer (FRET) between europium trisbipyridine cryptate (TBPEu(3+)) as energy donor and cross-linked allophycocyanin, symbolized by XL665, as acceptor, leading to a long-lived FRET signal. TBPEu(3+)-labeled uridine triphosphate (UTP), referred to as K-11-UTP in the text, was obtained by coupling TBPEu(3+) moiety to a C-5 functionalized UTP analog. K-11-UTP can be directly incorporated in RNA strands during enzymatic synthesis. This was demonstrated in an in vitro transcription reaction promoted by T(7) RNA polymerase. The reaction was performed in the presence of K-11-UTP and biotin-labeled cytidine triphosphate (biotin-16-CTP) in admixture with natural ribonucleotides. After the addition of streptavidin-XL665 conjugate (SA-XL665), which binds on biotinylated cytidine residues, a long-lived FRET signal was obtained. This proved that both europium cryptate and biotin were incorporated into the same RNA strand and are close enough to generate a FRET signal. The study of this FRET detection assay format showed that such doubly labeled RNA can be easily detected even when a very low percentage of K-11-UTP is used (less than 1% of total UTP concentration). Europium-cryptate-labeled RNA can also be monitored using a homogeneous hybridization assay format involving a biotinylated probe. After the addition of SA-XL665, the FRET signal generated demonstrates the formation of RNA:DNA hybrids. Europium-cryptate-labeled nucleotide thus gives access to a new type of RNA nonisotopic labeling and homogeneous detection assays.

Biotinylation↗
PubMed2000

Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding.

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates. The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.

Amino Acid Substitution↗
PubMed2001

Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.

Animals↗
PubMed2004

Protease-sensitive signalling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein.

The native cysteine residues of green fluorescent protein (GFP) at positions 48 and 70 were replaced by non-thiolic amino acids, and new cysteine sites were introduced at specific, surface positions. Based on molecular modeling of the GFP structure, the sites chosen for mutagenesis to Cys were glutamic acid at position 6 and isoleucine at position 229. These new, unique cysteine sites provided reactive thiol groups suitable for site-specific chemical modification by eosin-based fluorescence labels. The new constructs were designed to serve as the basis of proof of principle for fluorescence resonance energy transfer (FRET) using an enzyme-activated (trypsin) intervening sequence between native and chemically conjugated fluorophores. These eosin moieties provided chemical FRET partners for the native GFP chromophore. On excitation, these GFP-eosin constructs exhibited strong intramolecular FRET, with quenching of the native GFP (511 nm) fluorophore emission and emission around 540 nm, corresponding to eosin. GFP mutants engineered with trypsin-sensitive sequences close to the eosin site, so that on trypsinolysis FRET was destroyed, the emission wavelength switching from that of the chemical FRET partner back to that of the native GFP fluorophore, providing efficient, ratio-based detection. This protein engineering provides the basis for novel bioprobes for enzymatic triggering using intramolecular FRET between GFP and carefully sited chemical labels.

Binding Sites↗
PubMed2006

Assessment of prestin self-association using fluorescence resonance energy transfer.

An active process within the cochlea is necessary to obtain the sensitivity and frequency selectivity characteristic of mammalian hearing. This process is realized, at least in part, through the electromotile response of outer hair cells (OHCs). Electromotility requires the presence of prestin, a transmembrane protein highly expressed in the OHC lateral wall. Very little is known about how prestin functions at the molecular level to elicit electromotility, but theoretical models and recent experiments suggest that prestin-prestin interactions are required. To explore the extent of proposed prestin interactions, we employ fluorescence resonance energy transfer (FRET). FRET is a powerful optical technique capable of measuring inter-fluorophore distances less than 10 nm. Using human embryonic kidney cells (HEKs) as a model cell system and the standard FRET pair, cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor, we assay for the self-association of prestin under steady-state conditions using acceptor photobleach FRET (apFRET) and sensitized emission FRET (seFRET). Our findings from apFRET indicate the presence of prestin self-association when HEKs express both prestin-CFP and prestin-YFP in the membrane. The average FRET efficiency was approximately 9%, but values as high as 20% were measured. Notably, a higher efficiency of energy transfer ranging from 10-30% was obtained with seFRET. Additionally, we report an apFRET efficiency of approximately 10% for cells expressing a CFP-prestin-YFP double fusion protein. We discuss the significance of these measurements for establishing the presence of prestin-prestin interactions in transfected HEK cells.

Animals↗