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Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples.
PLoS One 2007; 2(10):e1011Plos

Abstract

BACKGROUND

Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET.

METHODOLOGY/PRINCIPAL FINDINGS

To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP.

CONCLUSIONS/SIGNIFICANCE

The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells.

Authors+Show Affiliations

Section of Molecular Cytology, Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. j.goedhart@science.uva.nlNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17925859

Citation

Goedhart, Joachim, et al. "Sensitive Detection of P65 Homodimers Using Red-shifted and Fluorescent Protein-based FRET Couples." PloS One, vol. 2, no. 10, 2007, pp. e1011.
Goedhart J, Vermeer JE, Adjobo-Hermans MJ, et al. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. PLoS ONE. 2007;2(10):e1011.
Goedhart, J., Vermeer, J. E., Adjobo-Hermans, M. J., van Weeren, L., & Gadella, T. W. (2007). Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. PloS One, 2(10), pp. e1011.
Goedhart J, et al. Sensitive Detection of P65 Homodimers Using Red-shifted and Fluorescent Protein-based FRET Couples. PLoS ONE. 2007 Oct 10;2(10):e1011. PubMed PMID: 17925859.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. AU - Goedhart,Joachim, AU - Vermeer,Joop E M, AU - Adjobo-Hermans,Merel J W, AU - van Weeren,Laura, AU - Gadella,Theodorus W J,Jr Y1 - 2007/10/10/ PY - 2007/08/22/received PY - 2007/09/20/accepted PY - 2007/10/11/pubmed PY - 2008/7/17/medline PY - 2007/10/11/entrez SP - e1011 EP - e1011 JF - PloS one JO - PLoS ONE VL - 2 IS - 10 N2 - BACKGROUND: Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/17925859/Sensitive_detection_of_p65_homodimers_using_red_shifted_and_fluorescent_protein_based_FRET_couples_ L2 - http://dx.plos.org/10.1371/journal.pone.0001011 DB - PRIME DP - Unbound Medicine ER -