在蛋白质和结构提炼中的胺和氨酸残留物的基组上进行气交换研究,使用与极侧链组的NOE限制物进行结构提炼
Mitsuhiro Takeda1, JunGoo Jee, Akira M Ono
1Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Journal of the American Chemical Society
|September 30, 2011
概括
新的核磁共振技术可以在氨酸和氨酸残留物中识别缓慢交换的基组. 这种进步有助于提炼蛋白质结构,因为它为基质子提供了重要的NOE限制.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 核磁共振 (NMR) 光谱学 核磁共振 (NMR) 光谱学
背景情况:
- 核磁共振 (NMR) 方法对于蛋白质结构的确定至关重要.
- 识别缓慢交换的极性侧链质子,如氨酸 (Tyr-OH) 和氨酸硫 (Cys-SH) 组,为蛋白质结构的精炼提供了有价值的核过量效应 (NOE) 约束.
- 之前的方法集中在Tyr-OH和Cys-SH组.
研究的目的:
- 扩展NMR方法,用于监测 (Ser) 和三 (Thr) (OH) 基的交换率.
- 为了证明由缓慢交换Ser/Thr-OH质子来获得的NOE限制的实用性,以精炼蛋白质结构.
- 在蛋白质EPPIb中识别具有缓慢交换OH组的特定Ser和Thr残留物.
主要方法:
- 在H(2) O/D(2) O (1:1) 混合物中使用了13C(β) -NMR信号监测.
- 使用的蛋白质在Ser/Thr残留的β碳上具有 (13) C, (2) H双标记.
- 分析了OH组的交换率,通过观察质子和化同位素的13C (β) 峰值的分辨率.
主要成果:
- 成功监测了18.2kDa蛋白质EPPIb中的Ser和ThrOH组的交换率.
- 在特定的缓冲条件下 (50mM酸盐,pH7.5,40°C) 在EPPIb中鉴定出一个Ser和四个Thr残留物,它们具有缓慢交换的基组 (k(ex) <~40s(-1)).
- 根据已识别的缓慢交换的Ser/Thr-OH组,收集了额外的NOE限制值.
结论:
- 扩展的NMR方法对于识别缓慢交换的Ser/Thr-OH组是有效的.
- 缓慢交换的Ser/Thr-OH质子为提炼蛋白质结构提供了宝贵的NOE限制.
- 这种方法在很大程度上有助于确定蛋白质中OH质子和结受体原子的空间位置.
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