Engineering of weak helper interactions for high-efficiency FRET probes.

TitleEngineering of weak helper interactions for high-efficiency FRET probes.
Publication TypeJournal Article
Year of Publication2013
AuthorsGrünberg, R, Burnier JV, Ferrar T, Beltran-Sastre V, Stricher F, van der Sloot AM, Garcia-Olivas R, Mallabiabarrena A, Sanjuan X, Zimmermann T, Serrano L
JournalNature methods
Date Published2013 Sep 1
Abstract

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.

DOI10.1038/nmeth.2625
Alternate JournalNat. Methods