使用自动投射光谱学 (APSY) 的蛋白质侧链共振的自动化NMR赋值
Sebastian Hiller1, Rosmarie Joss, Gerhard Wider
1Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
Journal of the American Chemical Society
|August 20, 2008
概括
本研究介绍了一种使用五维自动投影光谱 (5D APSY-NMR) 和ALASCA算法进行蛋白质侧链分配的自动化NMR方法. 这种技术有效地在小到中等球状蛋白质中赋予阿里法特共振.
科学领域:
- 结构生物学 结构生物学
- 生物物理学的生物物理.
- 核磁共振 (NMR) 光谱学 核磁共振 (NMR) 光谱学
背景情况:
- 蛋白质结构的确定依赖于NMR共振的准确分配.
- 侧链共振分配,特别是对异形部分,在NMR光谱学中是一个重大挑战.
- 现有的方法往往需要广泛的手工干预和专门的实验.
研究的目的:
- 开发和验证一种自动化方法,用于对异形蛋白侧链共振的序列特异性NMR赋值.
- 为了使小和中型球状蛋白质能够有效和准确地分配.
- 减少NMR数据分析所需的时间和专业知识.
主要方法:
- 记录一个五维 (5D) 自动投影光谱 (APSY-) NMR实验 (APSY-HC(CC-TOCSY) CONH).
- 利用 TOCSY 混合时间和投影角度的同时变化来捕获异形的 C-H 部分.
- 使用ALASCA (从APSY数据中局部和线性赋值侧链的算法) 算法分析APSY峰值列表.
主要成果:
- 阿拉斯加算法在20个常见的氨基酸侧链中实现了17个氨基酸侧链的明确分配,而剩下的3个则具有>95%的确定性.
- 在116残留蛋白TM1290上证明了可行性,实现了97%的碳和87%的质子共振检测和正确的分配.
- 5D APSY-HC ((CC-TOCSY) CONH实验成功地在光谱仪时间的24小时内分配了共振.
结论:
- 开发的自动化NMR方法为球状蛋白中的异形侧链分配提供了强大的和高效的方法.
- 预计该方法将常规适用于至少120个氨基酸的蛋白质.
- 这一进步大大简化了NMR分配过程,促进了结构研究.
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