Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy.
Two mutants of the blue copper protein azurin from Pseudomonas aeruginosa, Ile7Ser and Phe110Ser, were prepared. The mutations were aimed at affecting the mobility and the fluorescence properties of Trp48, the only tryptophan residue present, which in the wild-type protein is located in a highly hydrophobic and rigid environment. EPR, UV-vis, and NMR spectroscopy show that the copper binding site and the overall structure of the wild-type protein are preserved and that structural effects occur only on a local scale. Steady-state fluorescence spectra of both mutants, particularly in the copper-free form, show that tryptophan fluorescence is dramatically affected by the introduction of a polar residue close to it. The emission maximum is red-shifted and dependent on the excitation wavelength. This indicates a loosening of the matrix around the indolyl side chain and an increase of the effective dielectric constant of the microenvironment. Time-resolved fluorescence spectroscopy also shows substantial changes in the fluorescence lifetimes and in the distribution of the lifetimes of the mutants; these variations are interpreted in terms of a change in solvation of the Trp48 side chain.
Gilardi
G
G
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Mei
G
G
Rosato
N
N
Canters
G W
GW
Finazzi-Agrò
A
A
D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D007526:Isoelectric Point; D007532:Isoleucine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D012694:Serine; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*
D007532:Isoleucine; D001400:Azurin; D012694:Serine; D010649:Phenylalanine; D014364:Tryptophan