title: Time-resolved fluorescence study of azurin variants: conformational heterogeneity and tryptophan mobility.
abstract: Time-resolved fluorescence and time resolved fluorescence anisotropy studies have been performed on wild-type azurin from Pseudomonas aeruginosa and two variants to study the mobility of Trp48. The two azurin variants in which the microenvironment of Trp48 was changed comprised the single mutations Ile7Ser and Phe110Ser. The experiments were performed on the holo-Cu(I), holo-Cu(II), and apo- forms at various pH values, viscosities, and temperatures; two distinct parts of the emission spectrum were selected for detection. Two prominent subnanosecond lifetimes in the fluorescence decays of the Cu(II) proteins could be observed. The decay of apo-azurin also consists of more than one component. The occurrence of more than one component in the fluorescence decays is explained by conformational heterogeneity. The anisotropy decay results appeared to be different for wild-type and mutated azurins. Phe110Ser and Ile7Ser azurin show more mobility of the Trp48 residue, as reflected in the order parameter.
authors.affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands.
authors.forename: S J
authors.initials: SJ
authors.lastname: Kroes
authors.forename: G W
authors.initials: GW
authors.lastname: Canters
authors.forename: G
authors.initials: G
authors.lastname: Gilardi
authors.forename: A
authors.initials: A
authors.lastname: van Hoek
authors.forename: A J
authors.initials: AJ
authors.lastname: Visser
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D001400:Azurin; D014364:Tryptophan