在蛋白质工程和冷EM中的环形基因调节蛋白TRAP中,近邻合作的结构基础
Weicheng Li1, Haoyun Yang1,2, Kye Stachowski1
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210.
概括
托结合衰减蛋白 (TRAP) 调节基因表达. 工程TRAP变体揭示了连接体结合部位的接近如何驱动合作,影响RNA结合和基因调节.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 结构生物学 结构生物学
背景情况:
- 来自Alkalihalobacillus halodurans (Aha) 的托结合衰减蛋白 (TRAP) 是一种同型多多相蛋白,它调节了托生物合成.
- TRAP与托 (Trp) 连接体结合,随后与trp操作子mRNA领导序列结合,以降低基因表达的调节.
- 之前的研究表明,TRAP中存在合作性连接键结合,并怀疑近邻相互作用是该机制.
研究的目的:
- 调查负责TRAP连接体结合中的合作性的近邻相互作用.
- 设计和描述TRAP变体 (dTRAP) 来剖析合作机制.
- 为了阐明TRAP的全osteric调节的结构基础.
主要方法:
- 工程dTRAP变体与链接的子单元和突变的结合点.
- 使用核磁共振 (NMR),异热定位热量计 (ITC) 和本地质谱仪监测配体结合.
- 使用冷电子显微镜 (Cryo-EM) 的结构分析.
主要成果:
- 工程dTRAP变体与相邻的合格结合站点表现出强大的合作性.
- 将合格站点与突变站点分开导致了显著的绑定缺陷.
- 冷EM揭示了参与连接物和RNA结合的关闭环的连接物诱导稳定.
结论:
- 对于TRAP的合作托结合,近邻相互作用至关重要.
- 门环的结构动力学是TRAP的性调节的关键.
- 了解TRAP的合作性为特定的连接体敏感性提供了对全控制和蛋白质工程的洞察.
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