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Forster resonance energy transfer

Greta Faccio, Stefan Salentinig
Protein biosensors are widely used for the monitoring of metabolite concentration and enzymatic activities inside living cells and in in vitro applications. Neutrophil elastase (NE) is a serine protease of relevance in inflammatory diseases whose activity can lead to pathological conditions if unregulated. This study focuses on the structural characterization of a biosensor for NE activity based on Förster resonance energy transfer (FRET). The cleavage by NE results in dissociation of the FRET fluorescent protein pair and alteration of the fluorescent emission spectrum...
October 17, 2017: Biophysical Journal
James Joseph, Kevin N Baumann, Philipp Koehler, Tim J Zuehlsdorff, Daniel J Cole, Judith Weber, Sarah E Bohndiek, Silvia Hernández-Ainsa
Molecular rulers that rely on the Förster resonance energy transfer (FRET) mechanism are widely used to investigate dynamic molecular processes that occur on the nanometer scale. However, the capabilities of these fluorescence molecular rulers are fundamentally limited to shallow imaging depths by light scattering in biological samples. Photoacoustic tomography (PAT) has recently emerged as a high resolution modality for in vivo imaging, coupling optical excitation with ultrasound detection. In this paper, we report the capability of PAT to probe distance-dependent FRET at centimeter depths...
October 18, 2017: Nanoscale
Lichun Wei, Jiang Zhang, Zihao Mai, Fangfang Yang, Mengyan Du, Fangrui Lin, Junle Qu, Tongsheng Chen
Acceptor-sensitized quantitative Förster resonance energy transfer (FRET) measurement (E-FRET) is mainly impeded by donor emission crosstalk and acceptor direct excitation crosstalk. In this paper, we develop a novel E-FRET approach (Lux-E-FRET) based on linear unmixing (Lux) of the fluorescence intensity ratio between two detection channels with each excitation of two different wavelengths. The two detection channels need not to selectively collect the emission of donor or acceptor, and the excitation wavelengths need not to selectively excite donor or acceptor...
October 16, 2017: Optics Express
Ye Li, Xiaoning Song, Xiang Yi, Ruibing Wang, Simon Ming-Yuen Lee, Xueqing Wang, Ying Zheng
In the present study, the zebrafish was explored as an in vivo model to assess the biofate of transferrin receptor (TfR)-targeted coumarin 6 (C6) micelles across various biological barriers. Three 7peptide (7pep)-decorated poly-(ethylene glycol)-block-poly (ε-caprolactone) (PEG-b-PCL) micelles loaded with fluorescence coumarin 6 (7pep-M-C6) with different ligand densities were constructed with particle sizes between 30-40 nm. Whole-mount immunostaining revealed that the expression level of TfR in the retina, brain and intestine increased along with development stage...
October 17, 2017: ACS Applied Materials & Interfaces
Simin Belali, Ganapathi Emandi, Atillio A Cafolla, Barry O'Connell, Benjamin Haffner, Matthias E Möbius, Alireza Karimi, Mathias O Senge
3,5-Diformyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (3,5-diformyl-BODIPY) can be used as an efficient biofunctional cross-linker to generate a new class of chitosan-based hydrogels with fluorescence resonance energy transfer (FRET) dynamics and good solubility in water. The hydrogel was fully characterized by FT-IR, UV-vis, fluorescence, FE-SEM, AFM, rheology and picosecond time-resolved spectroscopic techniques. The self-healing ability was demonstrated by rheological recovery and macroscopic and microscopic observations...
October 17, 2017: Photochemical & Photobiological Sciences
Steven Daly, Luke MacAleese, Philippe Dugourd, Fabien Chirot
In the context of native mass spectrometry, the development of gas-phase structural probes sensitive to the different levels of structuration of biomolecular assemblies is necessary to push forward conformational studies. In this paper, we provide the first example of the combination of ion mobility (IM) and Förster resonance energy transfer (FRET) measurements within the same experimental setup. The possibility to obtain mass- and mobility-resolved FRET measurements is demonstrated on a model peptide and applied to monitor the collision-induced unfolding of ubiquitin...
October 16, 2017: Journal of the American Society for Mass Spectrometry
Kathrin Lehmann, Ruihan Zhang, Nathalie Schwarz, Alexander Gansen, Norbert Mücke, Jörg Langowski, Katalin Toth
Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear...
October 16, 2017: Scientific Reports
Dennis D Fernandes, Jasbir Bamrah, Senthilkumar Kailasam, Gregory-Neal W Gomes, Yuchong Li, Hans-Joachim Wieden, Claudiu C Gradinaru
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647)...
October 12, 2017: Scientific Reports
Jeremiah Traeger, Daniel K Schwartz
Single-molecule Förster Resonance Energy Transfer (FRET) was used to study the dynamic association of mobile donor-labeled ssDNA oligonucleotides ("target") with covalently immobilized complementary acceptor-labeled ssDNA oligonucleotides ("probe"). While probe-target association events were resolved for all experiments, such FRET events were far more likely to occur in systems with complementarity and on hydrophobic, as compared to hydrophilic, surfaces. The distribution of donor-acceptor association-time intervals did not exhibit simple first-order kinetics, and when decomposed into a superposition of first-order processes, only a small fraction of events corresponded to a long-lived state that was presumed to represent true DNA hybridization, while the majority of association events were transient, representing non-specific associations or partial hybridization...
October 12, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
Mayuri Sadoine, Michele Cerminara, Noémie Kempf, Michael Gerrits, Joerg Fitter, Alexandros Katranidis
Förster Resonance Energy Transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) which provides proteins with two different functional groups for posttranslational labeling at the specific amino acid positions...
October 12, 2017: Analytical Chemistry
Drew M Dolino, Sudeshna Chatterjee, David M MacLean, Charlotte Flatebo, Logan D C Bishop, Sana A Shaikh, Christy F Landes, Vasanthi Jayaraman
N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions...
October 9, 2017: Nature Chemical Biology
Zhaoyang Ding, Jinyun Tan, Gang Feng, Zhen Yuan, Changfeng Wu, Xuanjun Zhang
This work describes a facile yet powerful approach to energy-transfer NMOF (nanoscale metal-organic framework) fabrication for ratiometric peroxynitrite (ONOO(-)) sensing. Poly(vinyl alcohol) (PVA) is chosen to organize the energy donor (NMOF) and acceptor (molecular probes). PVA can conveniently graft onto the NMOF surface and bind to the molecular probes bearing the arylboronic acid group through multiple weak coordination interactions. Due to efficient Förster resonance energy transfer (FRET), the bright blue fluorescence of the NMOF is quenched while the green or red emission from the acceptor is enhanced...
July 1, 2017: Chemical Science
Jamuna K Vaishnav, Tushar Kanti Mukherjee
Herein we have developed a simple liposome-based donor-acceptor system across the lipid bilayer using 4',6-diamidino-2-phenylindole (DAPI) as the donor and an ultrasmall ligand-capped silver nanocluster (Ag NC) as the acceptor. The process of Förster resonance energy transfer (FRET) between DAPI and the Ag NC across the liposome bilayer has been demonstrated using steady-state and time-resolved fluorescence spectroscopy. The synthesized Ag NCs with a majority of Ag4 and Ag5 cores have been characterized using FTIR, mass spectrometry, HRTEM, UV-Vis and PL spectroscopy...
October 18, 2017: Physical Chemistry Chemical Physics: PCCP
Ben Shi, Xianfeng Gu, Zhijun Wang, Ge Xu, Qiang Fei, Jie Tang, Chunchang Zhao
Probes bearing good aqueous solubility and biocompatibility as well as fast response can serve as ideal tools for evaluating the underlying molecular mechanism of endogenous production of H2S caused by drugs; however, they are still lacking but highly desirable. Here, we demonstrate a novel strategy for constructing highly efficient H2S nanoprobes through locking Förster resonance energy transfer borondipyrromethene (BODIPY) pairs in water-dispersible core-shell silica nanoparticles. Importantly, these nanocomposites can effectively confine complementary guests within the same cores due to the existence of a shield, thus guaranteeing efficient Förster resonance energy transfer...
October 18, 2017: ACS Applied Materials & Interfaces
Sebastian Goetzl, Christian Teutloff, Tobias Werther, Sandra E Hennig, Jae-Hun Jeoung, Robert Bittl, Holger Dobbek
B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron-sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases...
October 17, 2017: Biochemistry
John J Ferrie, Naoya Ieda, Conor M Haney, Christopher R Walters, Itthipol Sungwienwong, Jimin Yoon, E James Petersson
Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range...
October 5, 2017: Chemical Communications: Chem Comm
Pia Ringer, Andreas Weißl, Anna-Lena Cost, Andrea Freikamp, Benedikt Sabass, Alexander Mehlich, Marc Tramier, Matthias Rief, Carsten Grashoff
Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3-5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy...
September 18, 2017: Nature Methods
Carlo R Bartoli, David Zhang, Jooeun Kang, Samson Hennessy-Strahs, David Restle, Jessica Howard, Gretchen Redline, Christian Bermudez, Pavan Atluri, Michael A Acker
BACKGROUND: Recent data suggest that hemolysis contributes to left ventricular assist device (LVAD) thrombosis, but the mechanism is unknown. In a clinical study, we measured plasma free hemoglobin (pfHgb) and the incidence of LVAD thrombosis. In an in vitro study, we examined biophysical relationships between shear stress, pfHgb and von Willebrand factor (vWF) metabolism toward understanding mechanisms of LVAD thrombosis. METHODS: In the clinical study, blood samples were obtained from continuous-flow LVAD patients (n = 30)...
September 21, 2017: Annals of Thoracic Surgery
Kathy R Chaurasiya, Remus T Dame
The complex binding dynamics between DNA and proteins are often obscured by ensemble averaging effects in conventional biochemical experiments. Single-molecule fluorescence methods are powerful tools to investigate DNA-protein interaction dynamics in real time. In this chapter, we focus on using single-molecule Förster Resonance Energy Transfer (smFRET) to probe the binding dynamics of individual proteins on single DNA molecules. We provide a detailed discussion of total internal reflection fluorescence (TIRF) instrument design, nucleic acid labeling with fluorophores, flow cell surface passivation, and data analysis methods...
2018: Methods in Molecular Biology
Siet M J L van den Wildenberg, Bram Prevo, Erwin J G Peterman
One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy...
2018: Methods in Molecular Biology
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