Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

ABSTRACT: Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics.

SUBMITTER: Naganbabu M

PROVIDER: S-EPMC4911959 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Fluorogenic Photoaffinity Labeling of Proteins in Living Cells.

Project description:Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive cross-linker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.

| S-EPMC6630177 | biostudies-literature

Rapid labeling of intracellular His-tagged proteins in living cells.

Project description:Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

| S-EPMC4364221 | biostudies-literature

Dynamic multicolor protein labeling in living cells.

Project description:Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST, hereafter called FAST) is a 14 kDa protein tag giving a bright green-yellow fluorescent complex upon interaction with the fluorogenic dye 4-hydroxy-3-methylbenzylidene rhodanine (HMBR). Here, we report a collection of fluorogens enabling tuning of the fluorescence color of FAST from green-yellow to orange and red. Beyond allowing the multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST's reversible labeling. This unprecedented behavior allows for selective detection of FAST-tagged proteins in cells expressing both green and red fluorescent species through two-color cross-correlation, opening up exciting prospects to overcome spectral crowding and push the frontiers of multiplexed imaging.

| S-EPMC5618792 | biostudies-literature

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

Project description:Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

| S-EPMC5100829 | biostudies-literature

Improved Photoinduced Fluorogenic Alkene-Tetrazole Reaction for Protein Labeling.

Project description:The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene-tetrazole reaction product. This finding provides useful guidance to the future design of alkene-tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene-tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene-tetrazole chemistry in real-time imaging in live cells.

| S-EPMC5688003 | biostudies-literature

Fluorogenic protein labeling using a genetically encoded unstrained alkene.

Project description:We developed a new fluorogenic bioorthogonal reaction that is based on the inverse electron-demand Diels-Alder reaction between styrene (an unstrained alkene) and a simple tetrazine. The reaction forms a new fluorophore with no literature precedent. We have identified an aminoacyl-tRNA synthetase/tRNA pair for the efficient and site-specific incorporation of a styrene-containing amino acid into proteins in response to amber nonsense codon. Fluorogenic labeling of purified proteins and intact proteins in live cells were demonstrated. The fluorogenicity of the styrene-tetrazine reaction can be potentially applied to the study of protein folding and function under physiological conditions with low background fluorescence interference.

| S-EPMC5369545 | biostudies-literature

HaloTag protein-mediated specific labeling of living cells with quantum dots.

Project description:Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging.

| S-EPMC2553894 | biostudies-literature

A fluorophore ligase for site-specific protein labeling inside living cells.

Project description:Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

| S-EPMC2890758 | biostudies-literature

?/?-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

Project description:Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-?-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both ?- and ?-amino acid residues ("?/?-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "?-peptides". This report documents an extension of the ?/?-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated ?/?-peptides based on a "stapled" Bim BH3 ?-peptide, which contains a hydrocarbon cross-link to enhance ?-helix stability. We show that a stapled ?/?-peptide can structurally and functionally mimic the parent stapled ?-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the ?/?-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled ?-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

| S-EPMC4687753 | biostudies-literature

Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling.

Project description:Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function.

| S-EPMC4414533 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data