敏感的核磁共振方法,以确定紧密结合的配体分子与蛋白质标的结合方式
Wan-Na Chen1, Christoph Nitsche1,2, Kala Bharath Pilla1
1Australian National University , Research School of Chemistry, Canberra, ACT 2601, Australia.
Journal of the American Chemical Society
|March 15, 2016
概括
这项研究引入了一种新型的核磁共振 (NMR) 方法,使用三甲基化学标签来确定具有挑战性的蛋白结合模式. 这种技术有助于在结晶不可能时以结构为导向的药物设计.
科学领域:
- 生物化学
- 结构生物学
- 药物发现
背景情况:
- 结构导向药物设计需要对蛋白质 - 连接体复合物的详细知识.
- 蛋白质 - 连接体共复合体的结晶通常具有挑战性或不可能.
- 现有的核磁共振 (NMR) 方法对研究密集结合体有局限性.
研究的目的:
- 开发一种敏感的NMR方法来确定具有难以向蛋白质的紧密结合化合物的结合模式.
- 克服目前依赖于快速配体交换或稳定同位素标记的NMR技术的局限性.
主要方法:
- 使用三基作为化学标签,利用其狭窄而强烈的1H NMR信号.
- 使用核重整效应光谱 (NOESY) 来产生内部和分子间的交叉峰值.
- 测量的伪接触从蛋白质上的化物标签转移到配体的位置.
- 证明了使用含有 tert-butyl 连接体的登革热病毒 NS2B-NS3 蛋白酶的方法.
主要成果:
- 甲基基组提供了非常强烈的分子内和分子间NOESY交叉峰值.
- 伪接触转移能够准确地定位连接物在蛋白质上.
- 在没有先前的蛋白质侧链共振分配的情况下实现了分子间NOE分配.
- 成功确定了与登革热病毒蛋白酶的高亲和性联体的结合模式.
结论:
- 提出的NMR方法对于确定难以实现的蛋白质标的联体结合模式具有敏感性和有效性.
- 这种方法提供了结晶的替代方案,克服了现有的NMR技术的局限性.
- 作为化学标签的 tert-butyl 组的使用促进了以结构为导向的药物设计,以挑战蛋白质 - 连接体系统.
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