Négrerie M, Gròf P, Bouet F, Ménez A, Aslanian D
Laboratoire de Physique des Solides, Université Pierre et Marie Curie, Paris, France.
Biochemistry. 1990 Sep 11;29(36):8258-65. doi: 10.1021/bi00488a009.
Raman spectroscopy was used to determine structural features of the native toxin alpha from Naja nigricollis, which contains only one Trp and one Tyr, and of chemically modified toxins having chromophores added to these two conserved aromatic amino acids. The percentages of secondary structure were determined by using amide I polypeptidic vibration analysis and are in agreement with X-ray structure [Low et al. (1976) Proc. Natl. Acad Sci. U.S.A. 73, 2991-2994] as well as with the geometry of the disulfide bridges estimated by using the v(S-S) vibrations. In the native toxin alpha, the single invariant tyrosine 25 appears to be buried in the structure and involved in a strong hydrogen bond. We have chemically modified these two invariant aromatic side chains by addition of chromophores. The presence of a (nitrophenyl)sulfenyl (NPS) chromophore bound to the Trp does not perturb the secondary structure of the toxin as shown by the analysis of the polypeptidic amide I vibrations; however, the environment of this Trp and the geometry of a disulfide bridge seem to be modified. The secondary structure is not affected by the presence of the NPS chromophore; therefore, the decrease in binding affinity observed after modification of Trp-29 by the reagent NPS-Cl [Faure et al. (1983) Biochemistry 22, 2068-2076] is due to an alteration of the environment of this aromatic amino acid and/or a steric hindrance and not to an overall modification of the toxin structure. The binding assays of [nitrotyrosyl]toxin show that after nitration the affinity toward the monoclonal antibody M alpha 1 is unchanged and that the affinity toward the cholinergic receptor (AcChR) from Torpedo marmorata remains high. We concluded that the structure of toxin alpha after adding the NO2 chromophore to Tyr-25 is the same as it is in native toxin.
拉曼光谱法被用于确定来自黑颈眼镜蛇的天然毒素α的结构特征，该毒素仅含有一个色氨酸和一个酪氨酸，还用于确定化学修饰毒素的结构特征，这些化学修饰毒素在这两个保守的芳香族氨基酸上添加了发色团。通过酰胺I多肽振动分析确定二级结构的百分比，其与X射线结构[洛等人（1976年）《美国国家科学院院刊》73，2991 - 2994]以及通过v(S - S)振动估计的二硫键几何结构一致。在天然毒素α中，单一不变的酪氨酸25似乎埋藏在结构中并参与一个强氢键。我们通过添加发色团对这两个不变的芳香族侧链进行了化学修饰。与色氨酸结合的（硝基苯基）亚磺酰基（NPS）发色团的存在，通过多肽酰胺I振动分析表明，并未扰乱毒素的二级结构；然而，这个色氨酸的环境以及一个二硫键的几何结构似乎发生了改变。二级结构不受NPS发色团存在的影响；因此，用试剂NPS - Cl修饰色氨酸 - 29后观察到的结合亲和力下降[福雷等人（1983年）《生物化学》22，2068 - 2076]是由于这个芳香族氨基酸环境的改变和/或空间位阻，而不是毒素结构的整体改变。[硝基酪氨酸]毒素的结合试验表明，硝化后对单克隆抗体Mα1的亲和力不变，并且对电鳐胆碱能受体（AcChR）的亲和力仍然很高。我们得出结论，在酪氨酸 - 25上添加NO2发色团后毒素α的结构与天然毒素相同。
