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.
Zauner
Gerhild
G
Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands.
Lonardi
Emanuela
E
Bubacco
Luigi
L
Aartsma
Thijs J
TJ
Canters
Gerard W
GW
Tepper
Armand W J W
AW
D000818:Animals; D003386:Brachyura / Q000737:chemistry; D003300:Copper / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008667:Metalloproteins / Q000737:chemistry*; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D011487:Protein Conformation; D013303:Streptomyces antibioticus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry*
D005456:Fluorescent Dyes; D008667:Metalloproteins; D003300:Copper; D014364:Tryptophan; D014442:Monophenol Monooxygenase; D010100:Oxygen