Monitoring Protein Interactions in the Living Cell Through the Fluorescence Decays of the Cyan Fluorescent Protein

Grailhe, Régis; Mérola, Fabienne; Ridard, J.; Couvignou, Stephen; Poupon, Chantal Le; Changeux, Jean‐Pierre; Laguitton-Pasquier, Hélène

doi:10.1002/cphc.200600057

Cited by 54 publications

(70 citation statements)

References 68 publications

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“…Our dimer construct resulted in a 38.5 Ϯ 1.5% FRET efficiency that falls in the range of expected values based on their molecular structure (supplemental Fig. S2A) (42) and corresponds very well to other reported FRET efficiencies for YFP-CFP dimer constructs that varied from 25 to 42% depending on the size of the linker (35,38,40,41,43). Łukasiewicz et al (39) reported an efficiency of 36% for a CFP-YFP dimer with a linker similar to the one we used.…”

Section: Discussionsupporting

confidence: 87%

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Bocksteins

Labro

Mayeur

et al. 2009

Journal of Biological Chemistry

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Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual ␣-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homoand heterotetrameric channels. Only the Kv2 ␣-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.Shaker-related channel subfamilies (Kv1-Kv6 and Kv8 -Kv9) have been identified based on the degree of sequence homology (1). Fully assembled Kv channels are composed of four ␣-subunits arranged around a central pore. Each ␣-subunit consists of six transmembrane segments S1-S6 with a cytoplasmic N and C terminus. The N terminus contains the T1 domain, a tetramerization domain that facilitates the assembly of ␣-subunits into functional channels. The presence of a T1 domain is not absolutely required for channel assembly because subunits without a T1 domain could also assemble into a functional tetramer, although less efficiently (2-4). However, the T1 domain not only promotes but also restricts the formation of possible homo-and heterotetramers by preventing incompatible subunits from assembling (5-7). When four compatible T1 domains assemble, they are arranged with the same 4-fold symmetry as the transmembrane segments, forming a hanging gondola structure (8).The T1 domain contains two subdomains, designated A and B box, which are both highly conserved within the different Kv subfamilies (9). Several residues in both the A and B boxes form the contact interface between neighboring subunits and are commonly assumed to be the key determinants for (subfamily specific) channel tetramer...

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Section: Discussionsupporting

confidence: 87%

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Bocksteins

Labro

Mayeur

et al. 2009

Journal of Biological Chemistry

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“…[26,30]. A recent report also comments on the necessity to fit 3 decay components to CFP-YFP bulk TCSPC data in cells, although the decay parameters were not reported [12]. In the absence of crystallographic evidence, we presume that the chromophore in Cerulean can adopt two conformations, similar to the CFP.…”

Section: Accepted M Manuscriptmentioning

confidence: 82%

“…due to tautomerisation, protonation or ion binding) mean that the fluorescence decay is often complex. Such dynamic complexity must be taken into consideration when extracting quantitative information from FRET measurements, and this is an emerging area of research [12][13][14][15][16].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

Millington

Grindlay

Altenbach

et al. 2007

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT High-Precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion proteinMichael Millington, [a, b] G. Joan Grindlay, [c] Kirsten Altenbach, [a, d] Robert K. Neely, [a, b] Walter Kolch, [ c, e] Mojca Bencina , [ f ] Nick D. Read, [a, d] Anita C. Jones, [a, b] David T. F.Dryden, [a, b]

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“…A 27-amino linker was used to connect the FPs CFP and YFP (15). The mCherry-YFP tandem was developed by substituting the CFP with the mCherry from the CFP-YFP construct.…”

Section: Constructionmentioning

confidence: 99%

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim

Kang

et al. 2011

Cytometry Pt A

Self Cite

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Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/ mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells. ' International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; two-photon spectral imaging; unmixing; protein-protein interaction; YFP; mCherry THE current advances in light microscopy and engineering of fluorescent proteins (FPs) with improved properties and altered colors have provided the cellular biologist with the ability to investigate fine molecular events in living cells (1,2). Properly combined, fluorescent molecules allow the study of protein conformational changes or inter protein interaction by means of fluorescence resonance energy transfer (FRET) (3,4). When an excited donor fluorophore is proximate (=10 nm) and suitably oriented to an acceptor molecule, FRET occurs through a nonradiative dipoledipole interaction. Currently, the most popular FP pairs for FRET analysis are cyan with yellow fluorescent protein (CFP/YFP) and green with red fluorescent protein (GFP/RFP) (5,6).Among the methodologies based on nonradiative resonance energy transfer, two approaches prevail, the fluorescence lifetime imaging microscopy (FLIM), and the fluorescence intensity-based FRET imaging. The FLIM is known to be cumbersome to require highly specialized equipment and as well to involve long acquisition time (7). To the contrary, fluorescence intensity-based FRET estimation provides

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Monitoring Protein Interactions in the Living Cell Through the Fluorescence Decays of the Cyan Fluorescent Protein

Cited by 54 publications

References 68 publications

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

Conserved Negative Charges in the N-terminal Tetramerization Domain Mediate Efficient Assembly of Kv2.1 and Kv2.1/Kv6.4 Channels

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

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