Tryptophan-to-Dye Fluorescence Energy Transfer Applied to Oxygen Sensing by Using Type-3 Copper Proteins

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. Oxygen makes the switch: We have devised a simple FRET‐based method to make O2‐binding “type‐3 copper” proteins, such as hemocyanin and tyrosinase, better suited for oxygen‐sensing applications. The endogenous tryptophan emission of these proteins reports on the binding of O2 to the copper centre, but this is difficult to detect in samples that show background fluorescence in the near‐UV region. To circumvent this problem, the tryptophan emission is translated to emission at higher wavelengths utilizing Fluorescence Resonance Energy Transfer to a label attached to the protein.