Monks S A, Gould A R, Lumley P E, Alewood P F, Kem W R, Goss N H, Norton R S
School of Biochemistry, University of New South Wales, Kensington, Australia.
Biochim Biophys Acta. 1994 Jul 20;1207(1):93-101. doi: 10.1016/0167-4838(94)90056-6.
The structure-function relationships of the neurotoxic polypeptide Sh I, from the sea anemone Stichodactyla helianthus, have been studied using limited proteolysis with trypsin and endoproteinase Lys-C. Major products from each of the proteolytic digests were characterised using N-terminal peptide sequencing and amino-acid analysis or mass spectrometry. Of the six possible tryptic cleavage sites in Sh I, the bonds adjacent to Arg-13 and Lys-47 were found to be the most susceptible, complete cleavage occurring within minutes. Cleavages adjacent to Lys-32 and Lys-46 proceeded more slowly and cleavage adjacent to Arg-45 was the slowest. The sixth potential site, adjacent to Lys-4, was not cleaved at all. All derivatives were inactive as crustacean neurotoxins. Cleavage with endoproteinase Lys-C generated two major products. Derivatives cleaved adjacent to Lys-32 and either Lys-46 or Lys-47 were isolated. Both were inactive, indicating that either cleavage adjacent to Lys-32 or the removal of the C-terminal lysine residue(s) was sufficient to abolish activity. Lys-4 again was refractory to cleavage. The sequence of cleavage events correlated well with the static accessibility of the lysyl and arginyl side chains and to a lesser extent with the accessibility of the carbonyl oxygen of susceptible peptide bonds, as measured from the solution structure of Sh I determined by 1H-NMR. In the case of Lys-4, the lack of cleavage by trypsin and endoproteinase Lys-C may reflect a lack of flexibility in this region. The effects of the various cleavages on biological activity emphasise that the surface of the protein near the reverse turn encompassing Asp-6, Asp-7 and Glu-8 is essential for activity.
利用胰蛋白酶和赖氨酸内切蛋白酶 Lys-C 进行有限蛋白水解，对来自海葵（赫氏列指海葵）的神经毒性多肽 Sh I 的结构 - 功能关系进行了研究。使用 N 端肽测序、氨基酸分析或质谱对每种蛋白水解消化产物的主要产物进行了表征。在 Sh I 的六个可能的胰蛋白酶切割位点中，发现与 Arg-13 和 Lys-47 相邻的键最易断裂，几分钟内即可完全切割。与 Lys-32 和 Lys-46 相邻的切割进行得较慢，与 Arg-45 相邻的切割最慢。第六个潜在位点，与 Lys-4 相邻，根本未被切割。所有衍生物作为甲壳类神经毒素均无活性。用赖氨酸内切蛋白酶 Lys-C 切割产生了两种主要产物。分离出在 Lys-32 以及 Lys-46 或 Lys-47 相邻处切割的衍生物。两者均无活性，这表明在 Lys-32 相邻处的切割或 C 端赖氨酸残基的去除足以消除活性。Lys-4 再次难以被切割。切割事件的顺序与赖氨酰和精氨酰侧链的静态可及性密切相关，在较小程度上与从通过 1H-NMR 确定的 Sh I 的溶液结构测量的敏感肽键的羰基氧的可及性相关。就 Lys-4 而言，胰蛋白酶和赖氨酸内切蛋白酶 Lys-C 未进行切割可能反映了该区域缺乏灵活性。各种切割对生物活性的影响强调，在包含 Asp-6、Asp-7 和 Glu-8 的反向转角附近的蛋白质表面对于活性至关重要。
