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Special Issue "Förster Resonance Energy Transfer (FRET) 2015"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Physical Chemistry, Theoretical and Computational Chemistry".

Deadline for manuscript submissions: closed (30 April 2015).

Special Issue Editor

Guest Editor
Prof. Dr. Herbert Schneckenburger

Institute of Applied Research, Aalen University, Beethovenstr. 1, 73430 Aalen, Germany
Website | E-Mail
Phone: +49 7361 576-3401
Interests: biomedical optics; microscopy; fluorescence; FRET

Special Issue Information

Dear Colleagues,

Förster resonance energy transfer (FRET) describes a non-radiative transfer of excitation energy from a donor to an acceptor molecule in the nanometre range. Although biological systems, e.g. photosynthetic organisms, have been using this mechanism for millions of years, it lasted until 1946, when Th. Förster described it theoretically. With the wide-spread use of fluorescent proteins in cell biology since the 1990’s, FRET experiments gained considerable importance for measurements of molecular conformations or interactions, even down to the single molecule level. This special volume is dedicated to the principles and applications of FRET ranging from model systems to living organisms.

Prof. Dr. Herbert Schneckenburger
Guest Editor

Submission

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Keywords

  • Förster resonance energy transfer (FRET)
  • fluorescence spectroscopy and microscopy
  • fluorescence lifetimes
  • living cells

Published Papers (14 papers)

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Research

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Open AccessArticle
Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)
Int. J. Mol. Sci. 2016, 17(3), 366; https://doi.org/10.3390/ijms17030366
Received: 6 November 2015 / Revised: 21 February 2016 / Accepted: 26 February 2016 / Published: 10 March 2016
Cited by 2 | PDF Full-text (2963 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Holliday Junctions are critical DNA intermediates central to double strand break repair and homologous recombination. The junctions can adopt two general forms: open and stacked-X, which are induced by protein or ion binding. In this work, fluorescence spectroscopy, metal ion luminescence and thermodynamic [...] Read more.
Holliday Junctions are critical DNA intermediates central to double strand break repair and homologous recombination. The junctions can adopt two general forms: open and stacked-X, which are induced by protein or ion binding. In this work, fluorescence spectroscopy, metal ion luminescence and thermodynamic measurements are used to elucidate the ion binding site and the mechanism of junction conformational change. Förster resonance energy transfer measurements of end-labeled junctions monitored junction conformation and ion binding affinity, and reported higher affinities for multi-valent ions. Thermodynamic measurements provided evidence for two classes of binding sites. The higher affinity ion-binding interaction is an enthalpy driven process with an apparent stoichiometry of 2.1 ± 0.2. As revealed by Eu3+ luminescence, this binding class is homogeneous, and results in slight dehydration of the ion with one direct coordination site to the junction. Luminescence resonance energy transfer experiments confirmed the presence of two ions and indicated they are 6–7 Å apart. These findings are in good agreement with previous molecular dynamics simulations, which identified two symmetrical regions of high ion density in the center of stacked junctions. These results support a model in which site-specific binding of two ions in close proximity is required for folding of DNA Holliday junctions into the stacked-X conformation. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics
Int. J. Mol. Sci. 2015, 16(8), 17637-17654; https://doi.org/10.3390/ijms160817637
Received: 27 March 2015 / Revised: 21 July 2015 / Accepted: 24 July 2015 / Published: 31 July 2015
Cited by 11 | PDF Full-text (1644 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the [...] Read more.
The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity
Int. J. Mol. Sci. 2015, 16(7), 16642-16654; https://doi.org/10.3390/ijms160716642
Received: 30 April 2015 / Revised: 30 June 2015 / Accepted: 6 July 2015 / Published: 22 July 2015
Cited by 2 | PDF Full-text (1518 KB) | HTML Full-text | XML Full-text
Abstract
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and [...] Read more.
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells
Int. J. Mol. Sci. 2015, 16(7), 14695-14716; https://doi.org/10.3390/ijms160714695
Received: 20 May 2015 / Revised: 15 June 2015 / Accepted: 17 June 2015 / Published: 30 June 2015
Cited by 20 | PDF Full-text (3060 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that [...] Read more.
Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Intravital FRET: Probing Cellular and Tissue Function in Vivo
Int. J. Mol. Sci. 2015, 16(5), 11713-11727; https://doi.org/10.3390/ijms160511713
Received: 20 February 2015 / Accepted: 13 May 2015 / Published: 21 May 2015
Cited by 12 | PDF Full-text (3618 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss [...] Read more.
The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Investigation of Förster Resonance Energy Transfer (FRET) and Competition of Fluorescent Dyes on DNA Microparticles
Int. J. Mol. Sci. 2015, 16(4), 7738-7747; https://doi.org/10.3390/ijms16047738
Received: 13 February 2015 / Revised: 27 March 2015 / Accepted: 27 March 2015 / Published: 8 April 2015
Cited by 1 | PDF Full-text (2879 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Fluorescent labeling is widely used to investigate the structural stability and changes to DNA nano- and microstructures. Despite this, the conventional method for observing DNA structures has several limitations in terms of cost-efficiency. This paper introduces a DNA spherical particle stained with DNA [...] Read more.
Fluorescent labeling is widely used to investigate the structural stability and changes to DNA nano- and microstructures. Despite this, the conventional method for observing DNA structures has several limitations in terms of cost-efficiency. This paper introduces a DNA spherical particle stained with DNA intercalating dyes (SYBR Green and SYTOX Orange) as tracers and reports the interaction between multiple dyes. The interference between the dyes was analyzed in terms of Förster resonance energy transfer (FRET) and competition. The changes in the fluorescence intensity by FRET were uniform, regardless of the sequence. The competition effect could occur when several dyes were added simultaneously. These properties are expected to help in the design of multicolor tracers in bioimaging and environmental applications. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
The CENP-T C-Terminus Is Exclusively Proximal to H3.1 and not to H3.2 or H3.3
Int. J. Mol. Sci. 2015, 16(3), 5839-5863; https://doi.org/10.3390/ijms16035839
Received: 20 January 2015 / Revised: 18 February 2015 / Accepted: 18 February 2015 / Published: 12 March 2015
Cited by 5 | PDF Full-text (2297 KB) | HTML Full-text | XML Full-text
Abstract
The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere–kinetochore complex contains specific nucleosomes and nucleosomal [...] Read more.
The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere–kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1C96A and H3.1C110A nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy
Int. J. Mol. Sci. 2015, 16(3), 5375-5385; https://doi.org/10.3390/ijms16035375
Received: 16 January 2015 / Revised: 12 February 2015 / Accepted: 26 February 2015 / Published: 9 March 2015
Cited by 10 | PDF Full-text (1544 KB) | HTML Full-text | XML Full-text
Abstract
Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet [...] Read more.
Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
Int. J. Mol. Sci. 2014, 15(12), 23836-23850; https://doi.org/10.3390/ijms151223836
Received: 28 October 2014 / Revised: 26 November 2014 / Accepted: 28 November 2014 / Published: 19 December 2014
Cited by 13 | PDF Full-text (804 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive [...] Read more.
Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation
Int. J. Mol. Sci. 2014, 15(10), 17827-17837; https://doi.org/10.3390/ijms151017827
Received: 22 July 2014 / Revised: 18 September 2014 / Accepted: 19 September 2014 / Published: 2 October 2014
Cited by 1 | PDF Full-text (388 KB) | HTML Full-text | XML Full-text
Abstract
Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose [...] Read more.
Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessArticle
Quantitative Analysis of Tau-Microtubule Interaction Using FRET
Int. J. Mol. Sci. 2014, 15(8), 14697-14714; https://doi.org/10.3390/ijms150814697
Received: 23 May 2014 / Revised: 30 June 2014 / Accepted: 14 July 2014 / Published: 21 August 2014
Cited by 6 | PDF Full-text (1494 KB) | HTML Full-text | XML Full-text
Abstract
The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using [...] Read more.
The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Review

Jump to: Research

Open AccessReview
Understanding FRET as a Research Tool for Cellular Studies
Int. J. Mol. Sci. 2015, 16(4), 6718-6756; https://doi.org/10.3390/ijms16046718
Received: 27 January 2015 / Accepted: 18 March 2015 / Published: 25 March 2015
Cited by 55 | PDF Full-text (1213 KB) | HTML Full-text | XML Full-text
Abstract
Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer [...] Read more.
Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessReview
Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques
Int. J. Mol. Sci. 2015, 16(3), 6076-6092; https://doi.org/10.3390/ijms16036076
Received: 3 February 2015 / Revised: 5 March 2015 / Accepted: 11 March 2015 / Published: 16 March 2015
Cited by 11 | PDF Full-text (1390 KB) | HTML Full-text | XML Full-text
Abstract
Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis [...] Read more.
Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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Open AccessReview
Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques
Int. J. Mol. Sci. 2014, 15(12), 22518-22538; https://doi.org/10.3390/ijms151222518
Received: 8 October 2014 / Revised: 8 November 2014 / Accepted: 18 November 2014 / Published: 5 December 2014
Cited by 170 | PDF Full-text (461 KB) | HTML Full-text | XML Full-text
Abstract
F resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional [...] Read more.
F resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λEX ∼ 280 nm, λEM ∼ 350 nm) , in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the proteinlocal environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic F resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins. Full article
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
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