Besse D, Budisa N, Karnbrock W, Minks C, Musiol H J, Pegoraro S, Siedler F, Weyher E, Moroder L
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Biol Chem. 1997 Mar-Apr;378(3-4):211-8. doi: 10.1515/bchm.1997.378.3-4.211.
Using methionine-auxotrophic Escherichia coli strains quantitative biosynthetic replacement of the methionine residues by seleno- and telluromethionine but not by methoxinine was achieved in various model proteins, clearly indicating a limited tolerance in the editing range of methionyl-tRNA synthetase. For expression of the protein variants the acetyl derivatives of the chalcogen-analogs of methionine, obtained by a new and highly efficient synthetic procedure, proved to be the ideal source in the growth media as they were found to be significantly more stable than the underivatized methionine analogs. The conformational properties in solution, the folding and unfolding parameters as well as X-ray crystallographic data confirmed the highly isomorphous character of the atomic mutants and thus the usefulness of this concept in X-ray analysis of proteins. Quantitative replacement of cysteine residues by selenocysteine has recently been achieved using cysteine-auxotrophic E. coli strains, but a selective replacement of cysteine residues by employing the natural translational machinery of selenocysteine is also conceivable. We have therefore performed a detailed study on synthetic selenocysteine-peptides in order to determine the redox potential of this cysteine analog, and thus the ability of related peptide and protein analogs to undergo the correct oxidative folding. Since the redox potential of selenocysteine was found to be significantly more reducing than that of the parent amino acid, selective formation of a diselenide bridge in presence of additional cysteine residues is highly favored as well documented in the case of the synthetic bis-selenocysteine-endothelin I analog. These results confirm that even cysteine residues may represent an interesting target for the design and expression of isomorphous heteroatomic analogs of proteins.
利用甲硫氨酸营养缺陷型大肠杆菌菌株，在各种模型蛋白中实现了用硒代甲硫氨酸和碲代甲硫氨酸对甲硫氨酸残基进行定量生物合成替代，但不能用甲氧基甲硫氨酸替代，这清楚地表明甲硫氨酰 - tRNA合成酶的编辑范围耐受性有限。对于蛋白变体的表达，通过一种新的高效合成方法获得的甲硫氨酸硫族元素类似物的乙酰衍生物，被证明是生长培养基中的理想来源，因为它们比未衍生化的甲硫氨酸类似物显著更稳定。溶液中的构象性质、折叠和去折叠参数以及X射线晶体学数据证实了原子突变体的高度同晶型特征，因此这一概念在蛋白质的X射线分析中是有用的。最近利用半胱氨酸营养缺陷型大肠杆菌菌株实现了用硒代半胱氨酸对半胱氨酸残基进行定量替代，但也可以设想利用硒代半胱氨酸的天然翻译机制选择性替代半胱氨酸残基。因此，我们对合成的硒代半胱氨酸肽进行了详细研究，以确定这种半胱氨酸类似物的氧化还原电位，从而确定相关肽和蛋白类似物进行正确氧化折叠的能力。由于发现硒代半胱氨酸的氧化还原电位比其母体氨基酸的氧化还原电位显著更负，在存在额外半胱氨酸残基的情况下，选择性形成二硒键是非常有利的，这在合成的双硒代半胱氨酸 - 内皮素I类似物的情况中得到了充分证明。这些结果证实，即使是半胱氨酸残基也可能是蛋白质同晶型杂原子类似物设计和表达的一个有趣靶点。
