Semisynthesis of Fluorescent Metabolite Sensors on Cell Surfaces

Brun, Matthias A.; Griss, Rudolf; Reymond, Luc; Tan, Kui‐Thong; Piguet, Joachim; Peters, Ruud J. R. W.; Vogel, Horst; Johnsson, Kai

doi:10.1021/ja206915m

Cited by 68 publications

(76 citation statements)

References 29 publications

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“…This behavior of Snifit-iGluR5 confirms the general validity of our sensor concept. 7 The maximum ratio change ΔR max of Snifit-iGluR5 amounts to 1.93 ± 0.11. By fitting the glutamate titration data to a single binding isotherm, we obtained a K d comp,glutamate = 12 ± 6 μM ( Figure 2B).…”

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confidence: 96%

“…We have shown in our previous work that the inclusion of a rigid polyproline linker between SNAP-and CLIP-tag leads to greatly improved sensor proteins in terms of their maximum ratio changes. 7 The polyproline linker increases the distance between the two fluorophores in the open state but not in the closed state of the sensor protein, which leads to a net increase of the sensor's dynamic range. 7 The fusion protein was successfully expressed and purified from Escherichia coli Rosetta gami and labeled with the molecules BG-PEG 11 -Cy5-glutamate 1 and CLIP-Surface 547 ( Figure S1B), which is a substrate for labeling CLIP-tag with the fluorescent dye DY-547 ( Figure S3).…”

mentioning

confidence: 99%

“…7 The polyproline linker increases the distance between the two fluorophores in the open state but not in the closed state of the sensor protein, which leads to a net increase of the sensor's dynamic range. 7 The fusion protein was successfully expressed and purified from Escherichia coli Rosetta gami and labeled with the molecules BG-PEG 11 -Cy5-glutamate 1 and CLIP-Surface 547 ( Figure S1B), which is a substrate for labeling CLIP-tag with the fluorescent dye DY-547 ( Figure S3). We found that the addition of 1 mM glutamate led to an increase of fluorescence emission of DY-547 and to a decrease of Cy5 fluorescence, indicating decreased FRET efficiency (Figure 2A).…”

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A Semisynthetic Fluorescent Sensor Protein for Glutamate

et al. 2012

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ABSTRACT:We report the semisynthesis of a fluorescent glutamate sensor protein on cell surfaces. Sensor excitation at 547 nm yields a glutamate-dependent emission spectrum between 550 and 700 nm that can be exploited for ratiometric sensing. On cells, the sensor displays a ratiometric change of 1.56. The high sensitivity toward glutamate concentration changes of the sensor and its exclusive extracellular localization make it an attractive tool for glutamate sensing in neurobiology.T he amino acid glutamate is the prevalent neurotransmitter in the vertebrate nervous system. It is used at well over 90% of the synapses in the human brain and influences essentially all forms of behavior, including consciousness, sensory perception, motor control, and mood. 1 Further, glutamate is involved at most synapses that are modifiable, that is, that are capable of adapting to changing patterns of stimuli by enhancing or reducing the efficiency of synaptic transmission. 2 These processes are thought to be responsible for high-order brain functions, such as learning and memory. Three fluorescent sensor proteins for investigating the role of glutamate in neurobiology have been developed so far. 3−5 However, the modest performance of these sensor proteins has limited their use. Here we present a semisynthetic fluorescent sensor protein for glutamate which shows higher sensitivity toward glutamate concentration changes and operates at longer wavelengths than the previously reported sensors.Semisynthetic fluorescent sensor proteins (Snifits), 6,7 are fusion proteins consisting of SNAP-tag, 8 CLIP-tag, 9 and an analyte-binding protein ( Figure 1A). SNAP-and CLIP-tag are labeled with a synthetic fluorescent ligand and a second synthetic fluorophore, respectively. The ligand binds to the binding protein in an intramolecular fashion and thereby keeps the sensor protein in a closed conformation. Free analyte can compete for binding to the binding protein and can shift the equilibrium to the open conformation. This shift can be detected by a change in the Forster resonance energy transfer (FRET) efficiency between the two fluorophores.For the construction of a glutamate sensor protein based on the Snifit sensor concept, we have chosen the ionotropic glutamate receptor 5 (iGluR5) as the binding protein for two reasons. First, due to the modular construction of ionotropic glutamate receptors, it is possible to express its glutamate binding domain S1S2 as a soluble protein in bacteria while conserving both the high affinity and specificity toward glutamate. 10,11 The possibility to characterize the soluble binding protein in vitro before its use on cell surfaces facilitates sensor development. Second, it is known that the stereoselective functionalization of the γ-carbon of the glutamate side chain does not significantly perturb its affinity toward iGluR5, 12,13 suggesting an attachment point for the required synthetic tether. We therefore prepared the tethered glutamate analogue 1 (Figure 1B, Scheme S1−S3) that contains a Cy5 fluorophore a...

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A Semisynthetic Fluorescent Sensor Protein for Glutamate

et al. 2012

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“…Small synthetic dyes generally give larger signal changes and may be more suitable for applications in vitro, whereas genetically encoded biosensors are the method of choice for measurements in cells or in vivo [9,10]. There are also approaches combining the two strategies by genetic fusion with protein tags, such as SNAP-tag or CLIP-tag [11,12], which can then be chemically modified with fluorescent dyes [13,14], as outlined in Sect. 2.3.…”

Section: Protein-based Biosensorsmentioning

confidence: 99%

Fluorescent Biosensors: Design and Application to Motor Proteins

Kunzelmann¹,

Solscheid²,

Webb³

2014

Experientia Supplementum

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Reagentless biosensors are single molecular species that report the concentration of a specific target analyte, while having minimal impact on the system being studied. This chapter reviews such biosensors with emphasis on the ones that use fluorescence as readout and can be used for real-time assays of concentration changes with reasonably high time resolution and sensitivity. Reagentless biosensors can be designed with different types of recognition elements, particularly specific binding proteins and nucleic acids, including aptamers. Different ways are described in which a fluorescence signal can be used to report the target concentration. These include the use of single, environmentally sensitive fluorophores; FRET pairs, often used in genetically encoded biosensors; and pairs of identical fluorophores that undergo reversible stacking interactions to change fluorescence intensity. The applications of these biosensors in different types of real-time assays with motor proteins are described together with some specific examples. These encompass regulation and mechanism of motor proteins, using both steady-state assays and single-turnover measurements.

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“…Some breakthroughs occurred in the covalent bio-conjugate field, where the same target ligand for capture may be chemically coupled with unique color fluorophores, a very similar approach to using commercially labeled antibodies for labeling cells (Chen et al, 2007;Kosaka et al, 2009;Vivero-Pol et al, 2005;Lee et al, 2010;Liu et al, 2014;Uttamapinant et al, 2010;Wombacher et al, 2010;Yao et al, 2012). Likewise, other groups have utilized bio-conjugate platforms based on tandem dye interactions that have resulted in fluorescence resonance energy transfer (FRET), a donor-acceptor approach that amplifies the Stokes shift of a molecule resulting in fluorescence emissions at longer wavelengths (Brun et al, 2009(Brun et al, , 2011Gallo et al, 2015;Pham et al, 2015;Rajapakse et al, 2010;Robers et al, 2009;Saunders et al, 2014;Yushchenko et al, 2012;Zürn et al, 2010). As a result, we find that current methods prove lacking in multi-color detection and real-time signal modulation.…”

Section: Introductionmentioning

confidence: 99%

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

Gallo

Jarvik

2017

Journal of Cell Science

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A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multiselectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cellimpermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity -displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via colabeling, and assess real-time cell surface receptor traffic via pulsechase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.

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Semisynthesis of Fluorescent Metabolite Sensors on Cell Surfaces

Cited by 68 publications

References 29 publications

A Semisynthetic Fluorescent Sensor Protein for Glutamate

A Semisynthetic Fluorescent Sensor Protein for Glutamate

Fluorescent Biosensors: Design and Application to Motor Proteins

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

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