Gilardi G, Mei G, Rosato N, Canters G W, Finazzi-Agrò A
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Biochemistry. 1994 Feb 15;33(6):1425-32. doi: 10.1021/bi00172a020.
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.
制备了来自铜绿假单胞菌的蓝色铜蛋白天青蛋白的两个突变体，即Ile7Ser和Phe110Ser。这些突变旨在影响唯一存在的色氨酸残基Trp48的迁移率和荧光特性，在野生型蛋白中，Trp48位于高度疏水且刚性的环境中。电子顺磁共振（EPR）、紫外可见光谱和核磁共振（NMR）光谱表明，野生型蛋白的铜结合位点和整体结构得以保留，且结构效应仅在局部尺度上发生。两个突变体的稳态荧光光谱，特别是无铜形式的，表明色氨酸荧光受到靠近它引入的极性残基的显著影响。发射最大值发生红移且依赖于激发波长。这表明吲哚侧链周围的基质松弛以及微环境的有效介电常数增加。时间分辨荧光光谱还显示突变体的荧光寿命和寿命分布有实质性变化；这些变化根据Trp48侧链溶剂化的变化来解释。
