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Proceedings Paper

Multipurpose indicator in cells using intramolecular FRET between GFP and chemical compounds
Author(s): Miho Suzuki; Yoichiro Ito; Hannah Elizabeth Savage; Yuzuru Husimi; Kenneth T. Douglas
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Paper Abstract

Specific, surface cysteine sites have been introduced into Green Fluorescent Protein (GFP) to allow site-specific chemical modification by thiol-directed reagents. These sites have been labelled using BODIPY/eosin/rhodamine reagents as chemical FRET partners for the native GFP chromophore. When they were excited at 488 nm these engineered GFP: conjugated-fluorophore constructs, showed quenching of the native GFP fluorophore emission at 511 nm. New emission bands appeared correponding to each chemical fluorophore emission. Thus the new GFP chimeras exhibited strong intramolecular FRET. GFP mutants were then engineered with trypsin-sensitive sequences located close to the chemical fluorophore-bearing cysteine site. Trypsinolysis caused major changes in the FRET fluorescence spectra. On trypsinolysis the FRET was destroyed, as the FRET partners were now on separate molecules in the cleaved products. T Consequently, the emission wavelength altered from that of the chemically conjugated FRET partner back to that of the native fluorophore of the GFP (511 nm). This provides the possibility of efficient, ratio-based detection. Thus, protein re-engineering has led to novel probes capable of enzymatic triggering based on intramolecular FRET between GFP and specifically sited chemical labels.

Paper Details

Date Published: 12 September 2003
PDF: 10 pages
Proc. SPIE 4967, Genetically Engineered and Optical Probes for Biomedical Applications, (12 September 2003); doi: 10.1117/12.477872
Show Author Affiliations
Miho Suzuki, Saitama Univ. (Japan)
Yoichiro Ito, Osaka Univ. (Japan)
Hannah Elizabeth Savage, Univ. of Manchester (United Kingdom)
Yuzuru Husimi, Saitama Univ. (Japan)
Kenneth T. Douglas, Univ. of Manchester (United Kingdom)

Published in SPIE Proceedings Vol. 4967:
Genetically Engineered and Optical Probes for Biomedical Applications
Darryl J. Bornhop; Ramesh Raghavachari; Samuel I. Achilefu; Alexander P. Savitsky, Editor(s)

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