Engineering of weak helper interactions for high-efficiency FRET probes

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.

The Efficacy of Raf Kinase Recruitment to the GTPase H-ras Depends on H-ras Membrane Conformer-specific Nanoclustering

“…Although we are using full-length proteins or protein domains, we nevertheless employed the monomeric variant of enhanced GFP, termed mGFP, to minimize the chances of tag-induced binding artifacts in the membrane context. The effect of enhanced fluorescent protein interaction on FRET in proteinprotein interaction studies was recently systematically addressed (60). The objective was to increase the FRET by either enhancing the "stickiness" of fluorescent proteins or even to introduce helper interactions.…”

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone

“…15,16 To bring NanoLuc and mNeonGreen in close proximity and ensure efficient BRET in the absence of antibody, we introduced so-called helper domains, a strategy that was recently introduced by Serrano and coworkers for FRET sensors. 27 The interaction between the helper domains should be strong enough to keep the sensor in a compact state in the absence of an antibody, but easily disrupted upon antibody binding. Ideally, helper domains consist of peptides or compact, easilyfolded small protein domains whose affinity can be easily tuned.…”

Engineering Genetically Encoded FRET Sensors

“…Longer linkers, while minimizing interference of the PPI, result in only a very weak FRET signal due to the distance dependence of FRET. Recently, Serrano and colleagues provided a solution to this problem, by using a peptide-domain interaction as a secondary interaction for the fluorescent domains [98]. The peptide (Wp2) was fused adjacent to one FP, while a small domain (WW) with affinity for the peptide was fused adjacent to the other fluorescent domain (Figure 6A).…”

“…The peptide-domain interaction (from a set of previously characterized pairs) was chosen so that the interaction occurred only in the presence of a primary PPI. This “helper” interaction, with a K d of around 170 μM, improved the detection of PPIs by fluorescence lifetime imaging, by increasing FRET efficiencies about two-fold, from ∼20% to ∼40% [98]. The same helper domains were also used to improve the dynamic range of a caspase FRET sensor consisting of mTurquoise2 and mCitrine.…”