Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors

Ai, Hui‐wang; Hazelwood, Kristin L.; Davidson, Michael W.; Campbell, Robert E.

doi:10.1038/nmeth.1207

Cited by 327 publications

(292 citation statements)

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“…Single FP-type biosensors are better suited to multiparameter imaging than FRET-based biosensors which each require two distinct hues of FP. 20 With the goal of eventually producing an expanded repertoire of fluorescent hues for FP-based biosensors, we have undertaken the development of circularly permuted monomeric red FPs (RFPs). 21 Our starting template is the engineered variant of Discosoma RFP 22 known as mCherry.…”

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confidence: 99%

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

2010

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Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein-based biosensors for Ca 21 and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)-based biosensors by providing a means for varying the critical dipole-dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th b-strand of mCherry. These new variants may ultimately prove useful for the creation of single FP-based Ca 21 biosensors.

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confidence: 99%

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

2010

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“…Commonly, FRET is measured by the fluorescence intensity ratio of the acceptor to the donor. In that case, whatever the two fluorescent protein FRET pairs chosen, CFP/YFP and mOrange/mCherry [42], mTFP1/mCitrine and mAmetrine/tdTomato [43,44], mTagBFP/sfGFP and mVenus/ mKok [45], the multiplex approach suffers from two limitations: (i) a spectral bleed-through of the first acceptor in the second donor emission band that depends directly on the respective quantities of the two biosensors and (ii) the multiple excitation wavelength which requires sequential acquisition that does not adequately follow fast signal dynamics or signal changes in highly motile samples.…”

Section: New Methodological Insights For Multiplexing Kinase Biosensorsmentioning

confidence: 99%

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Sizaire¹

2017

Protein Phosphorylation

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Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system.

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“…For the specific case of genetically encoded FRET-based biosensors, this limitation is even more restricting for two reasons: first because each biosensor requires two FPs, and second because most FPs have relatively broad excitation and emission spectra, although this can be partly alleviated with appropriate excitation and emission filters. Despite this, coimaging two FRET pairs that utilize four spectrally distinct FPs has been demonstrated recently (38). As a result, researchers can now apply a number of coimaging techniques, and in some cases a combination of such techniques, to coimage two or more signaling events in the same cell (39).…”

Section: Challenges Associated With Coimagingmentioning

confidence: 99%

“…2B), coimaging two FRET pairs that use distinct donors and a common acceptor (40) (Fig. 2C), and utilizing FPs that have unique excitation and emission properties that make them suitable as FRET partners yet distinguishable from the other FPs in a filter-based setup (38,39) (Fig. 2D).…”

Section: Coimaging Fret-based Biosensors Via Spectrally Distinct Fluomentioning

confidence: 99%

“…For example, Ai et al engineered a GFP mutant, mAmetrine, with a long Stokes-shift, or the shift between fluorophore absorption and emission maxima, that was used to obtain two distinct coimageable FRET pairs (38). Specifically, mAmetrine was found to be a suitable FRET donor for the orange fluorescent protein (OFP), tdTomato, and this FRET pair was coimaged with another FRET pair using the monomeric teal FP, mTFP1, as the energy donor for the acceptor YFP mCitrine.…”

Section: Coimaging Fret-based Biosensors Via Spectrally Distinct Fluomentioning

confidence: 99%

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Dynamic visualization of signal transduction in living cells: From second messengers to kinases

Herbst

Zhang

2009

IUBMB Life

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SummaryThe study of signal transduction, or the highly regulated series of biochemical events which allow a cell to convert a given stimulus into a functional response, has seen a paradigm shift with a recent explosion in the number of genetically encoded FRET-based biosensors capable of detecting spatial and temporal regulation of various signaling events in living cells. The two classes of biosensors discussed, namely kinase activity and second messenger biosensors, utilize two fluorescent proteins (FP) suitable for FRET and convert a signaling event of interest into a conformational change in the biosensor that can be measured as a change in FRET between the two FPs. Individually, these biosensors have been used to elucidate many complex signal transduction mechanisms in various biological systems. However, it has become increasingly clear that it is often more desirable to study multiple signaling events simultaneously, allowing for precise correlation of the temporal profiles of multiple signaling molecules without the complication of cell to cell variability. With the design of spectrally distinct biosensors and new coimaging strategies, simultaneous imaging of multiple signaling events is not only possible, but has aided in mapping the intricate network of cellular signal transduction cascades. Furthermore, as aberrant signal transduction involving second messengers and kinases is implicated in numerous disease states, it is hopeful that these FRET-based biosensors and coimaging strategies can help to unravel the molecular links between altered signal transduction and certain disease states.2009 IUBMB IUBMB Life, 61(9): 902-908, 2009

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Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors

Cited by 327 publications

References 11 publications

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

Dynamic visualization of signal transduction in living cells: From second messengers to kinases

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