title: 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.
authors.affiliation: 1] EMBL/CRG Systems Biology Research Unit, Center for Genomic Regulation, Barcelona, Spain. [2] Pompeu Fabra University, Barcelona, Spain. [3] Institut de Recherche en Immunologie et en Cancérologie, Université de Montréal, Montréal, Canada.
authors.forename: Raik
authors.initials: R
authors.lastname: Grünberg
authors.forename: Julia V
authors.initials: JV
authors.lastname: Burnier
authors.forename: Tony
authors.initials: T
authors.lastname: Ferrar
authors.forename: Violeta
authors.initials: V
authors.lastname: Beltran-Sastre
authors.forename: François
authors.initials: F
authors.lastname: Stricher
authors.forename: Almer M
authors.initials: AM
authors.lastname: van der Sloot
authors.forename: Raquel
authors.initials: R
authors.lastname: Garcia-Olivas
authors.forename: Arrate
authors.initials: A
authors.lastname: Mallabiabarrena
authors.forename: Xavier
authors.initials: X
authors.lastname: Sanjuan
authors.forename: Timo
authors.initials: T
authors.lastname: Zimmermann
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D055672:Static Electricity; D048490:raf Kinases / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D048490:raf Kinases; D018631:ras Proteins; D000097573:Red Fluorescent Protein; C434479:citrine protein, bacteria