title: Protease-sensitive signalling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein.
abstract: The native cysteine residues of green fluorescent protein (GFP) at positions 48 and 70 were replaced by non-thiolic amino acids, and new cysteine sites were introduced at specific, surface positions. Based on molecular modeling of the GFP structure, the sites chosen for mutagenesis to Cys were glutamic acid at position 6 and isoleucine at position 229. These new, unique cysteine sites provided reactive thiol groups suitable for site-specific chemical modification by eosin-based fluorescence labels. The new constructs were designed to serve as the basis of proof of principle for fluorescence resonance energy transfer (FRET) using an enzyme-activated (trypsin) intervening sequence between native and chemically conjugated fluorophores. These eosin moieties provided chemical FRET partners for the native GFP chromophore. On excitation, these GFP-eosin constructs exhibited strong intramolecular FRET, with quenching of the native GFP (511 nm) fluorophore emission and emission around 540 nm, corresponding to eosin. GFP mutants engineered with trypsin-sensitive sequences close to the eosin site, so that on trypsinolysis FRET was destroyed, the emission wavelength switching from that of the chemical FRET partner back to that of the native GFP fluorophore, providing efficient, ratio-based detection. This protein engineering provides the basis for novel bioprobes for enzymatic triggering using intramolecular FRET between GFP and carefully sited chemical labels.
authors.affiliation: Department of Functional Materials Science, Faculty of Engineering, Saitama University, 255 Shimo-okubo, Saitama, Saitama 338-8570, Japan. miho@fms.saitama-u.ac.jp
authors.forename: Miho
authors.initials: M
authors.lastname: Suzuki
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Ito
authors.forename: Hannah Elizabeth
authors.initials: HE
authors.lastname: Savage
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Douglas
mesh_terms: D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism*; D004801:Eosine Yellowish-(YS) / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014357:Trypsin / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; D014357:Trypsin; D003545:Cysteine; D004801:Eosine Yellowish-(YS)