Time-resolved fluorescence study of azurin variants: Conformational heterogeneity and tryptophan mobility

被引:41
作者
Kroes, SJ
Canters, GW
Gilardi, G
van Hoek, A
Visser, AJWG
机构
[1] Leiden Univ, Leiden Inst Chem, Gorlaeus Labs, NL-2300 RA Leiden, Netherlands
[2] Univ London Imperial Coll Sci Technol & Med, London SW7 2AZ, England
[3] Agr Univ Wageningen, Dept Biomol Sci, Microspect Ctr, Biochem Lab, NL-6703 HA Wageningen, Netherlands
关键词
D O I
10.1016/S0006-3495(98)77688-X
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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 Trp(48). The two azurin variants in which the microenvironment of Trp(48) was changed comprised the single mutations Ile(7)Ser and Phe(110)Ser. 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 it; explained by conformational heterogeneity. The anisotropy decay results appeared to be different for wild-type and mutated azurins, Phe(110)Ser and Ile(7)Ser azurin show more mobility of the Trp(48) residue, as reflected in the order parameter.
引用
收藏
页码:2441 / 2450
页数:10
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