对RAS家族小GTPase蛋白DiRAS3的生物化学和生物物理表征
Takashi Saitoh1, Ha-Neul Kim2, Riku Narita3
1Faculty of Pharmaceutical Sciences, Hokkaido University of Science, Sapporo, Hokkaido, 006-8585, Japan; Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, M5G 1L7, Canada.
Protein expression and purification
|August 31, 2023
概括
DiRAS3,一种抑制瘤的小GTPase,表现出独特的生化特性. 它的N-和C-终端延伸介导弱多元化,影响其核酸结合状态,尽管改变了G-box动图.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 结构生物学 结构生物学
背景情况:
- 迪拉斯3 (ARHI) 是RAS亚家族的小GTPase,具有瘤抑制功能,与RAS原瘤基因不同.
- 它的生物化学和生物物理特性由于不稳定性和溶解度低,因此仍然不太了解.
- 了解DiRAS3的结构功能关系对于其生物学作用至关重要.
研究的目的:
- 描述DiRAS3.3的生物化学和生物物理特性.
- 研究N和C终端扩展在DiRAS3稳定性,溶解性和多元化中的作用.
- 为了确定DiRAS3的核酸结合能力.
主要方法:
- 设计了一种可溶的DiRAS3变体 (C75S/C80S),用于大肠杆菌的复合表达.
- 使用Ni-NTA和尺寸排除染色学 (SEC) 净化复合DiRAS3.
- 产生了N-终端 (ΔN) 和N-和C-终端 (ΔNC) 截断结构;使用SEC和1H-15N HSQC NMR进行分析.
主要成果:
- 改进的DiRAS3变种显示出更好的溶解性和表达.
- 证券交易委员会分析显示,全长的DiRAS3存在于单体-二元平衡状态,而ΔNC是单体.
- ΔN构造表现出聚合的单体行为; ΔNC构造表现出分散的NMR峰值,表明一个功能性的GTPase域能够结合GDP/GTP.
结论:
- 迪拉斯3具有功能性GTPase域,能够结合核酸并切换状态,尽管存在G盒式基因替代.
- N-和C-终端扩展在DiRAS3的二分化和聚合中起作用.
- 这些扩展与GTPase域暂时相互作用,调解DiRAS3.3的弱多元化.
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