Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.

译文

:山羊α-乳清蛋白(GLA)包含四个色氨酸(Trp)残基。为了获得有关天然GLA中单个Trp残基的荧光贡献的信息,我们记录了四个GLA突变体W26F,W60F,W104F和W118F的荧光光谱,其中每个单个Trp残基都被苯丙氨酸替代了( Phe)。四个突变体与野生型GLA的荧光光谱比较表明,在天然GLA中,三个Trp残基(Trp60,Trp104和Trp118)被强烈淬灭,并说明了Trp26的部分间接淬灭。结果,野生型GLA和突变体W60F,W104F和W118F的荧光主要来自于Trp26。晶体结构的检查表明,除了与Trp60和Trp118的吲哚基团直接接触的二硫键外,与Trp60,Trp104和Trp118的吲哚基团直接接触的主链肽键也有贡献。直接淬火的效果。有趣的是,缺乏对Trp26的直接淬灭解释了为什么紫外光对二硫键的裂解更多地由高荧光的Trp26而不是由较少荧光的Trp104和Trp118介导。

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