Solving the α-Conotoxin Folding Problem: Efficient Selenium-Directed On-Resin Generation of More Potent and Stable Nicotinic Acetylcholine Receptor Antagonists
Solving the α-Conotoxin Folding Problem: Efficient Selenium-Directed On-Resin Generation of More Potent and Stable Nicotinic Acetylcholine Receptor Antagonists
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DOI：
10.1021/ja910602h
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发表时间：
2010-03-17
影响因子：
15
通讯作者：
Alewood, Paul F.
中科院分区：
文献类型：
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作者：
Muttenthaler, Markus;Nevin, Simon T.;Alewood, Paul F.
α-芋螺毒素是紧密折叠的小蛋白，其拮抗烟碱乙酰胆碱受体（nAChR），对不同亚型具有高特异性。在这里，我们报告了使用硒代半胱氨酸在支持相的方法，以直接天然折叠和生产α-芋螺毒素有效地改善生物物理特性。通过在两亲性树脂上用硒代半胱氨酸对替换互补的半胱氨酸对，我们能够化学地将α-芋螺毒素的所有五个结构亚类专门引导到它们的天然折叠中。α-硒芋螺毒素PnIA在1.4埃分辨率下的X射线分析证实了二硒键的电子等排特征和α-芋螺毒素折叠的完整性。α-硒芋螺毒素在大鼠膈肌和非洲爪蟾卵母细胞中表达的α 3 β 4、α 7和α 1 β 1 δ γ nAChR中表现出相似或改善的效力，加上在血浆中改善的二硫键混乱稳定性。总之，这些结果支持开发更稳定和有效的烟碱拮抗剂，适用于新的药物治疗，并强调硒代半胱氨酸技术的应用更广泛的二硫键合的肽和蛋白质。
alpha-Conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 angstrom resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha 3 beta 4, alpha 7, and alpha 1 beta 1 delta gamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.