Homogeneity and variability in the structure of azurin molecules studied by fluorescence decay and circular polarization.
The fluorescence decay of apoazurin derived from Pseudomonas aeruginosa is monoexponential. By this criterion the population of molecules of apoazurin is homogeneous. The emission anisotropy factor and the absorption anisotropy factor at the red edge of the absorption band assume similar values, showing that the tryptophan residue in apoazurin has the same asymmetric environment both in the ground and excited states. This finding suggests tight packing of the protein at the tryptophan environment. Native azurin does not decay monoexponentially. Moreover, comparison between the quantum yield calculated from the decay kinetics and the one measured directly shows that the majority of the azurin molecules are not fluorescent. There is thus variability in the structure of azurin molecules with an equilibration time that is longer than the fluorescence lifetime. Different asymmetric environment was found for the tryptophan residue in oxidized and reduced holoprotein and in apoazurin, as studied by the circular polarization of the fluorescence. D(2)O increases the fluorescence lifetime of apoazurin by 6 percent, compared to the lifetime in H(2)O solution; therefore water molecules may have access to the tryptophan residue, though the latter is situated in a hydrophobic environment.
D000577:Amides; D001059:Apoproteins; D001400:Azurin; D001426:Bacterial Proteins; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000032:analysis; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis
D000577:Amides; D001059:Apoproteins; D001426:Bacterial Proteins; D001400:Azurin; D014364:Tryptophan; D003903:Deuterium