FRET‐based binding assay between a fluorescent <scp>cAMP</scp> analogue and a cyclic nucleotide‐binding domain tagged with a CFP

Romero, Francisco Aguilar; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

doi:10.1002/1873-3468.12760

Cited by 3 publications

(17 citation statements)

References 26 publications

(43 reference statements)

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“…We call mAmetrine1.2 just mAmetrine in this manuscript for simplicity. The plasmid, pRSETB-EPAC1 CNBD -CFP, was previously prepared in our group [1]. Cyclic AMP analogs 8-Fluo-cAMP, 8-(2-[DY-547]aminoethylthio)adenosine-3,5-cyclic monophosphate (8-[DY-547]-AET-cAMP), and 8-CPT-cAMP were acquired from BioLog Life Science Institute (Bremen, Germany) through Axxora LLC.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we developed an intermolecular FRET-based binding assay for cAMP using cyan fluorescent protein (CFP) fused to the cyclic-nucleotide binding domain of EPAC1 (EPAC1 CNBD -CFP) as donor and fluorescein-labeled cAMP (8-(2-[Fluoresceinyl]aminoethylthio)adenosine-3,5-cyclic monophosphate, 8-Fluo-cAMP) (Supplementary Figure 1) as acceptor [1]. This FRET-based binding assay is more sensitive than other fluorescence-based methods such as fluorescence anisotropy [2] or fluorescence enhancement using the environment-sensitive fluorophore 8-(2-[7-Nitro-4-benzofurazanyl]aminoethylthio)adenosine-3,5-cyclic monophosphate (8-NBD-cAMP) [3].…”

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confidence: 99%

“…This FRET-based binding assay is more sensitive than other fluorescence-based methods such as fluorescence anisotropy [2] or fluorescence enhancement using the environment-sensitive fluorophore 8-(2-[7-Nitro-4-benzofurazanyl]aminoethylthio)adenosine-3,5-cyclic monophosphate (8-NBD-cAMP) [3]. The high sensitivity of the FRET-based binding assay is due to the direct measurement of the fluorescence intensity of the bright donor (or the acceptor) being employed [1]. …”

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confidence: 99%

“…Namely, the excitation light of CFP (donor) significantly excites 8-Fluo-cAMP independently of FRET. In consequence, a part of 8-Fluo-cAMP emission is produced by direct excitation not only mediated through FRET [1]. Therefore, a simple fluorescence spectrum of the mixture of EPAC1 CNBD -CFP and 8-Fluo-cAMP cannot tell us reliably if FRET occurs or not.…”

mentioning

confidence: 99%

“…Therefore, a simple fluorescence spectrum of the mixture of EPAC1 CNBD -CFP and 8-Fluo-cAMP cannot tell us reliably if FRET occurs or not. To determine the degree of FRET between EPAC1 CNBD -CFP and 8-Fluo-cAMP, we measured the fluorescence spectra in the absence and presence of excess amount of nonfluorescent cAMP analog (8-(4-Chlorophenylthio)adenosine-3,5-cyclic monophosphate [8-CPT-cAMP]) (Supplementary Figure 1), and determined the difference of the donor fluorescence intensities between the two conditions [1]. It is also possible to confirm FRET by photobleaching of the acceptor with intensive illumination and subsequent fluorescence recovery of the donor [4], although it is difficult to determine precisely the degree of FRET with this method.…”

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confidence: 99%

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Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

et al. 2018

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Fluorescence (or Frster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.

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confidence: 99%

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Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

et al. 2018

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High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga

Kinoshita-Terauchi

Shiba

Terauchi

et al. 2019

Zygote

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SummaryMale gamete chemotaxis towards the female gamete is a general strategy to facilitate the sexual reproduction in many marine eukaryotes. Biochemical studies of chemoattractants for male gametes of brown algae have advanced in the 1970s and 1980s, but the molecular mechanism of male gamete responses to the attractants remains elusive. In sea urchin, a K+ channel called the tetraKCNG channel plays a fundamental role in sperm chemotaxis and inhibition of K+ efflux through this channel by high K+ seawater blocks almost all cell responses to the chemoattractant. This signalling mechanism could be conserved in marine invertebrates as tetraKCNG channels are conserved in the marine invertebrates that exhibit sperm chemotaxis. We confirmed that high K+ seawater also inhibited sperm chemotaxis in ascidian, Ciona intestinalis (robusta), in this study. Conversely, the male gamete chemotaxis towards the female gamete of a brown alga, Mutimo cylindricus, was preserved even in high K+ seawater. This result indicates that none of the K+ channels is essential for male gamete chemotaxis in the brown alga, suggesting that the signalling mechanism for chemotaxis in this brown alga is quite different from that of marine invertebrates. Correlated to this result, we revealed that the channels previously proposed as homologues of tetraKCNG in brown algae have a distinct domain composition from that of the tetraKCNG. Namely, one of them possesses two repeats of the six transmembrane segments (diKCNG) instead of four. The structural analysis suggests that diKCNG is a cyclic nucleotide-modulated and/or voltage-gated K+ channel.

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cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins

2021

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Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders. For example, modulation of cAMP-mediated intracellular signaling pathways by anti-tumor drugs could reduce tumor growth. However, most early stage tools used for measuring the cAMP level in living organisms require cell disruption, which is not appropriate for live cell imaging or animal imaging. Thus, in the last decades, tools were developed for real-time monitoring of cAMP distribution or signaling dynamics in a non-invasive manner. Genetically-encoded sensors based on fluorescent proteins and luciferases could be powerful tools to overcome these drawbacks. In this review, we discuss the recent genetically-encoded cAMP sensors advances, based on single fluorescent protein (FP), Föster resonance energy transfer (FRET), single luciferase, and bioluminescence resonance energy transfer (BRET) for real-time non-invasive imaging.

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FRET‐based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide‐binding domain tagged with a CFP

Cited by 3 publications

References 26 publications

Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

Robust Evaluation of Intermolecular FRET Using a Large Stokes Shift Fluorophore as a Donor

High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga

cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins

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