Tryptophan-to-dye fluorescence energy transfer applied to oxygen sensing by using type-3 copper proteins |
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Authors: | Zauner Gerhild Lonardi Emanuela Bubacco Luigi Aartsma Thijs J Canters Gerard W Tepper Armand W J W |
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Affiliation: | Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands. |
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Abstract: | A fluorescence-based system to sense oxygen in solution is described. The method exploits the sensitivity of the endogenous fluorescence of type-3 copper proteins towards the presence of oxygen by translating the near-UV emission of the protein to label fluorescence in the visible range through a FRET mechanism. The main protein in this study, a recombinant tyrosinase from the soil bacterium Streptomyces antibioticus, has been covalently labeled with a variety of fluorescent dye molecules with emission maxima spanning the whole visible wavelength range. In all cases, the emission of the label varied considerably between O2-bound and O2-free protein with a contrast exceeding that of the Trp emission for some labels. It is shown that different constructs may be simultaneously observed using a single excitation wavelength. Next to the described application in oxygen sensing, the method may be applicable to any protein showing variations in tryptophan fluorescence, for example as a function of ligand binding or catalysis. |
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Keywords: | copper fluorescence sensors oxygen proteins tryptophan |
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