Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples.
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.


RESULTS
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
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.
Goedhart
Joachim
J
Section of Molecular Cytology, Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. j.goedhart@science.uva.nl
Vermeer
Joop E M
JE
Adjobo-Hermans
Merel J W
MJ
van Weeren
Laura
L
Gadella
Theodorus W J
TW
D001426:Bacterial Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D015233:Models, Statistical; D038761:Photobleaching; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods; D051996:Transcription Factor RelA / Q000737:chemistry*; D000097573:Red Fluorescent Protein
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051996:Transcription Factor RelA; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria