Quantitative assessment of fluorescent proteins

Cranfill, Paula J.; Sell, Brittney R.; Baird, Michelle A.; Allen, John R.; Lavagnino, Zeno; Gruiter, H. Martijn de; Kremers, Gert‐Jan; Davidson, Michael W.; Ustione, Alessandro; Piston, David W.

doi:10.1038/nmeth.3891

Cited by 458 publications

(536 citation statements)

References 65 publications

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“…Understanding this effect will be critical for designing RFPs with high fluorescence yields, especially in light of the low brightness of most RFPs with large Stokes shifts. 21 Although our results emphasize the importance of the acylimine region for the Stokes shift, hydrogen-bonding to the chromophore p-hydroxyphenyl group also impacts the fluorescence properties. We also examined these interactions in our MD simulations of TagRFP675, mKate/M41Q, and mKate and find they are very similar.…”

mentioning

confidence: 60%

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675

Konold

Yoon

Lee

et al. 2016

J. Phys. Chem. Lett.

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Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally-resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show ps timescale red-shifts followed by ns time blue-shifts. Global analysis of the TRF spectra reveals spectrally-distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

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mentioning

confidence: 60%

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675

Konold

Yoon

Lee

et al. 2016

J. Phys. Chem. Lett.

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“…Furthermore, even half-size fluorescent protein (VC) could compensate for the defect of SMS dimerization by truncation of the C-terminal tail and lead to restored ER export (data not shown). Fluorescent proteins provide a very useful experimental tool; however, they sometimes alter protein functional behavior and subcellular localization because of their relatively large molecular mass and dimer formation (45,46). We reckon that augmented interaction by dimerization of the fluorescent proteins increases the stability of homodimer of SMSs, leading to restored ER export.…”

Section: C-terminal Tails Of Smss Are Involved In Homodimer Stabilitymentioning

confidence: 99%

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum

Hayashi

Nemoto-Sasaki

Matsumoto

et al. 2017

Journal of Biological Chemistry

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Edited by Dennis R. VoelkerSphingomyelin synthase (SMS) is the key enzyme for crosstalk between bioactive sphingolipids and glycerolipids. In mammals, SMS consists of two isoforms: SMS1 is localized in the Golgi apparatus, whereas SMS2 is localized in both the Golgi and plasma membranes. SMS2 seems to exert cellular functions through protein-protein interactions; however, the existence and functions of quaternary structures of SMS1 and SMS2 remain unclear. Here we demonstrate that both SMS1 and SMS2 form homodimers. The SMSs have six membrane-spanning domains, and the N and C termini of both proteins face the cytosolic side of the Golgi apparatus. Chemical cross-linking and bimolecular fluorescence complementation revealed that the N-and/or C-terminal tails of the SMSs were in close proximity to those of the other SMS in the homodimer. Homodimer formation was significantly decreased by C-terminal truncations, SMS1-⌬C22 and SMS2-⌬C30, indicating that the C-terminal tails of the SMSs are primarily responsible for homodimer formation. Moreover, immunoprecipitation using deletion mutants revealed that the C-terminal tail of SMS2 mainly interacted with the C-terminal tail of its homodimer partner, whereas the C-terminal tail of SMS1 mainly interacted with a site other than the C-terminal tail of its homodimer partner. Interestingly, homodimer formation occurred in the endoplasmic reticulum (ER) membrane before trafficking to the Golgi apparatus. Reduced homodimerization caused by C-terminal truncations of SMSs significantly reduced ER-to-Golgi transport. Our findings suggest that the C-terminal tails of SMSs are involved in homodimer formation, which is required for efficient transport from the ER.

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“…With the availability of brighter, more photostable fluorophores that have a reduced propensity to oligomerise (Cranfill et al, 2016) and have distinct structural features (e.g. electrostatic charge), we wished to determine whether an alternate fluorophore may allow us to develop a more accurate Akt reporter.…”

Section: Introductionmentioning

confidence: 99%

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Norris

Yang

Krycer

et al. 2017

Journal of Cell Science

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Akt is a key node in a range of signal transduction cascades and play a critical role in diseases such as cancer and diabetes. Fluorescentlytagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T-Akt2 over eGFP-Akt2 enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information.This article has an associated First Person interview with the first author of the paper.

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Quantitative assessment of fluorescent proteins

Cited by 458 publications

References 65 publications

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

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