固态NMR化学转移分析用于确定与Na,K-ATPase结合的ATP在其原生膜中的构造
David A Middleton1, John Griffin1, Mikael Esmann2
1Department of Chemistry, Lancaster University Bailrigg Lancaster LA1 4YB UK d.middleton@lancaster.ac.uk +44 (0)1524 594328.
RSC advances
|November 30, 2023
概括
魔法角旋转固态NMR (SSNMR) 揭示了与Na,K-ATPase结合的腺三酸盐 (ATP) 的核糖构造. 这种技术提供了有关原生膜内的性联体蛋白相互作用的关键细节.
科学领域:
- 生物物理学的生物物理.
- 结构生物学 结构生物学
- 生物化学 生物化学
背景情况:
- 确定与膜蛋白结合的小,反应性连接体的结构,与X射线结晶学和冷电子显微镜等传统方法具有挑战性.
- 不稳定的连接物经常在这些技术的样本准备过程中失去它们的原生构造或脱离.
- 了解连接体构造对于阐明蛋白质功能和药物设计至关重要.
研究的目的:
- 为了证明13C的实用性,化学物质从神奇角度旋转固态NMR (MAS SSNMR) 转移到在膜蛋白中表征可变联体构造.
- 在其原生膜环境中与Na,K-ATPase (NKA) 结合时,研究腺三酸盐 (ATP) 的核糖构成.
- 建立MAS SSNMR作为研究-蛋白相互作用细节的强大工具.
主要方法:
- 使用冷捕获来稳定膜蛋白和连接体的不稳定复合体.
- 使用MAS SSNMR对冷的蛋白质-连接体复合体进行13C化学转移的测量.
- 采用密度函数理论 (DFT) 计算,将实验化学转移与特定的ATP结构相关联.
- 执行二极合测量以确定H-C-C-H扭转角和完善结构模型.
主要成果:
- 13C MAS SSNMR成功地解决了与NKA的高亲和复合体中的ATP核糖和腺因碳的信号.
- 与DFT计算相比,实验性化学转移表明,在E1Na状态下,与NKA结合的ATP具有C3'-endo (N) 核糖构造.
- 这与在E2P状态下观察到的ATP和在E1复合体中观察到的AMPPCP中观察到的C2'-endo (S) 核糖体构造形成鲜明对比.
- 额外的测量证实了核糖环和氨酸环的相对方向.
结论:
- 马斯SSNMR,特别是C13化学转移,提供了独特和有价值的信息,在功能性膜蛋白内可变联体的构成.
- 该研究成功确定了与NKA结合的ATP的特定核糖构造,为酶机制提供了洞察力.
- 这种方法提供了一种强大的,但尚未充分利用的方法,用于在原生膜环境中详细分析连接体-蛋白相互作用.
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