de Antonio C, Martínez del Pozo A, Mancheño J M, Oñaderra M, Lacadena J, Martínez-Ruiz A, Pérez-Cañadillas J M, Bruix M, Gavilanes J G
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Proteins. 2000 Nov 15;41(3):350-61. doi: 10.1002/1097-0134(20001115)41:3<350::aid-prot70>3.0.co;2-v.
alpha-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called alpha-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 degrees C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9 degrees C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein-vesicle interaction.
α-肌动蛋白是一种来自巨大曲霉的强效细胞毒性蛋白，在第4位和第51位含有两个色氨酸残基。已经制备并纯化得到了两种单突变体W4F和W51F以及双突变体W4/51F，使其达到均一状态。这两个残基对于该蛋白在核糖体上的高度特异性核糖核酸酶活性（产生所谓的α片段）以及与脂质膜的相互作用（囊泡的聚集和融合）都不是必需的，尽管涉及色氨酸-51的突变形式显示核糖核酸酶活性降低。质子核磁共振数据表明，用苯丙氨酸取代色氨酸-51后，该酶的整体结构没有发生显著变化。每个色氨酸残基的取代都会导致热变性中点下降4℃，双突变体的中点则低9℃。色氨酸-51负责该蛋白大部分的近紫外圆二色性，并且也对肽键区域中该蛋白的整体椭圆率有贡献。色氨酸-51不显示荧光发射。膜结合蛋白比相应的游离形式在更低的温度下发生热变性。该蛋白与磷脂双层的相互作用促进了色氨酸-51量子产率的大幅增加，并且其荧光发射被掺入此类双层疏水区域的蒽淬灭。这表明在蛋白与囊泡相互作用后，该残基周围的区域位于双层的疏水核心中。
