The oxidation state of a protein observed molecule-by-molecule.
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.
Schmauder
Ralf
R
Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands. schmidt@physics.leidenuniv.nl
Librizzi
Fabio
F
Canters
Gerard W
GW
Schmidt
Thomas
T
Aartsma
Thijs J
TJ
D000042:Absorption; D001400:Azurin / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013499:Surface Properties
D002232:Carbocyanines; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D001400:Azurin; D003300:Copper