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Engineering of weak helper interactions for high-efficiency FRET probes


Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

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Figure 1: Design of FRET helper interactions.
Figure 2: Enhanced FRET.
Figure 3: Application of conventional and hiFRET probes to the study of H-Ras–Raf1 signaling.
Figure 4: Detection of a transient Raf1–B-Raf interaction induced by a B-Raf kinase inhibitor.

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This work was supported by the Human Frontiers Science Program (LT-fellowship to R.G.) and the European Union (projects EMERGENCE and PROSPECTS, grant agreement number HEALTH-F4-2008-201648, to L.S.) as well as the Spanish Ministry of Education and Science (Juan de la Cierva fellowship to A.M.v.d.S.). We thank M. Therrien for suggesting the B-Raf–Raf1 detection as well as M. Tyers for supporting the project.

Author information

Authors and Affiliations



R.G. and L.S. conceived the study. R.G. designed experiments and synthetic genes. F.S., R.G., A.M.v.d.S. and L.S. performed modeling and FoldX calculations. R.G. and T.F. performed in vitro experiments. J.V.B. and V.B.-S. performed cell culture experiments. R.G.-O., A.M., X.S., J.V.B. and V.B.-S. measured and analyzed FLIM. T.Z. and R.G. further analyzed FLIM data. X.S. and V.B.-S. performed caspase experiments. R.G., L.S., J.V.B., and T.Z. wrote the paper.

Corresponding authors

Correspondence to Raik Grünberg or Luis Serrano.

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Competing interests

Methods described in this study are subject to two patent applications (PCT/EP2012/075737 and PCT/EP2012/075743) filed by some of the authors (R.G., L.S. and F.S).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Tables 1–6 and Supplementary Notes 1–4 (PDF 3706 kb)

Supplementary Data 1

Annotated sequences of synthetic proteins and sensors constructs (GenBank format) (ZIP 75 kb)

Supplementary Data 2

In vitro FRET efficiencies (complete data set of FRET measurements on purified proteins) (XLS 45 kb)

Supplementary Software

Python scripts for selection of mutations and cell trace plotting (ZIP 5 kb)

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Grünberg, R., Burnier, J., Ferrar, T. et al. Engineering of weak helper interactions for high-efficiency FRET probes. Nat Methods 10, 1021–1027 (2013).

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