6533b7d7fe1ef96bd12685f4
RESEARCH PRODUCT
The oxidation state of a protein observed molecole-by-molecule.
Ralf SchmauderGerard W. CantersThijs J. AartsmaFabio LibrizziThomas Schmidtsubject
Surface PropertiesChemistryFluorescence spectrometryFluorescence correlation spectroscopyCarbocyaninesFluorescence in the life sciencesPhotochemistrySensitivity and SpecificityFluorescenceAtomic and Molecular Physics and OpticsFluorescence spectroscopyAbsorptionSpectrometry FluorescenceResonance fluorescenceAzurinPseudomonas aeruginosaFluorescence Resonance Energy TransferFluorescence cross-correlation spectroscopyPhysical and Theoretical ChemistryAzurinOxidation-ReductionCopperFluorescent Dyesdescription
We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectroscopy (FCS) combined with fluorescence intensity distribution analysis (FIDA), and on surfaces by using wide-field fluorescence microscopy.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2005-07-02 |