In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag

Miller, Leon K.; Cai, Yunfei; Sheetz, Michael P.; Cornish, Virginia W.

doi:10.1038/nmeth749

Cited by 278 publications

(239 citation statements)

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“…Heterodimeric conjugates of the common antibiotic trimethoprim (TMP) covalently linked to various terbium chelates were developed that bind noncovalently to eDHFR. TMP and eDHFR interact exclusively with one another in mammalian systems, and this interaction has been previously exploited to develop TMP-fluorophore conjugates that specifically label eDHFR fusion proteins in a variety of wild-type mammalian cell lines (LigandLink™ Universal Labeling Technology, Active Motif, Inc.) (33)(34)(35). In our previous study, we found that one particular conjugate, TMP-Lumi4 ( Fig.…”

Section: Resultsmentioning

confidence: 96%

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Rajapakse

Gahlaut

Mohandessi

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

133

114

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Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of proteinprotein interactions. A luminescent terbium complex, TMP-Lumi4, was introduced into cultured cells using two methods: (i) osmotic lysis of pinocytic vesicles; and (ii) reversible membrane permeabilization with streptolysin O. Upon intracellular delivery, the complex was observed to bind specifically and stably to transgenically expressed Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins. LRET between the eDHFR-bound terbium complex and green fluorescent protein (GFP) was detected as long-lifetime, sensitized GFP emission. Background signals from cellular autofluorescence and directly excited GFP fluorescence were effectively eliminated by imposing a time delay (10 μs) between excitation and detection. Background elimination made it possible to detect interactions between the first PDZ domain of ZO-1 (fused to eDHFR) and the C-terminal YV motif of claudin-1 (fused to GFP) in single microscope images at subsecond time scales. We observed a highly significant (P < 10 −6 ), six-fold difference between the mean, donornormalized LRET signal from cells expressing interacting fusion proteins and from control cells expressing noninteracting mutants. The results show that time-resolved LRET microscopy with a selectively targeted, luminescent terbium protein label affords improved speed and sensitivity over conventional FRET methods for a variety of live-cell imaging and screening applications. cellular imaging | dihydrofolate reductase | Forster resonance energy transfer | lanthanide luminescence | protein labeling P rotein-protein interactions, often mediated by modular interaction domains, play a fundamental role in the dynamic organization of cells (1). Various experimental techniques such as immunoprecipitation, affinity chromatography, and yeast twohybrid analysis have been used to identify putatively interacting proteins and deduce the biomolecular mechanisms of cell function (2, 3). However, cell-free studies and screening assays do not provide information about the spatio-temporal organization of protein networks in the natural environment of the living cell or organism. A variety of optical methods are available for monitoring protein interactions in cells, including fluorescence cross correlation spectroscopy (FCCS) (4, 5), bimolecular fluorescence complementation (6), translocation-based assays (7-9), and methods that detect intermolecular Förster resonance energy transfer (FRET). Among these methods, only FRET allows dynamic and reversible imaging of protein-protein interactions while simultaneously preserving information about their subcellular distribut...

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Section: Resultsmentioning

confidence: 96%

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Rajapakse

Gahlaut

Mohandessi

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

133

114

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“…To overcome this limitation for further applications, methotrexate was substituted with the antibiotic trimethoprim (TMP). TMP binds to eDHFR with high affinity (1 nM K D ) but exhibits minimal binding to mammalian DHFR (K D > 1 mM) [17]. It has been demonstrated that trimethoprim conjugates can be used to selectively label eDHFR in wild type mammalian cells.…”

Section: Noncovalent Protein Tagsmentioning

confidence: 99%

“…The 18 kDa monomeric eDHFR behaves well when expressed in mammalian cells, and TMP-fluorophore conjugates with standard linker and protection group chemistry show good cell permeability. Labeling of nuclear proteins, plasma membrane proteins, and cytoplasmic proteins have been demonstrated in various cell types, showing that the eDHFR-TMP receptor-ligand pair can be used as a robust noncovalent protein labeling system in living cells [17][18]. The TMP-tag is marketed as LigandLink by Active Motif (Carlsbad, CA).…”

Section: Noncovalent Protein Tagsmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…Two types of technology stand out for their high performance. The first is a series of 'protein-directed' labeling techniques including DHFR [28], SNAP-tag [29], CLIP-tag [29], and Halo-tag [30]-based conjugations. The conjugations between these proteins and their substrates exhibit high selectivity and high affinity, even within cells and cell extracts [24].…”

Section: Box 1 Labeling Proteins With Organic Fluorophores Within Celmentioning

confidence: 99%

Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag

Cited by 278 publications

References 17 publications

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Time-resolved luminescence resonance energy transfer imaging of protein–protein interactions in living cells

Chemical tags: Applications in live cell fluorescence imaging

Bringing single-molecule spectroscopy to macromolecular protein complexes

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