一个基因编码的旋转标签用于电子磁共振距离测量
Moritz J Schmidt1, Julia Borbas, Malte Drescher
1Department of Chemistry, Zukunftskolleg, and Konstanz Research School Chemical Biology, University of Konstanz , Universitätsstraße 10, 78457 Konstanz, Germany.
Journal of the American Chemical Society
|January 17, 2014
概括
研究人员在大肠杆菌中基因编码了一个自旋标记的氨基酸,使得自旋标记蛋白质在没有化学步骤的情况下直接在细胞内产生用于电子磁共振 (EPR) 研究的蛋白质.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 生物物理学的生物物理.
背景情况:
- 电子磁共振 (EPR) 光谱是一种研究蛋白质动态和结构的强大技术.
- 传统的EPR研究需要对蛋白质进行化学标记,这可能是复杂的,可能会改变蛋白质的功能.
- 有需要的方法,允许直接,特定地点的标签的蛋白质在活细胞内.
研究的目的:
- 在大肠杆菌 (E. coli) 中基因编码一种新型,自旋标记的氨基酸.
- 为了使自旋标记蛋白质的细胞内生物合成,消除了化学标记的需要.
- 探索这种方法对细胞内EPR研究和分子内距离测量的实用性.
主要方法:
- 在大肠杆菌中非正规氨基酸的遗传编码.
- 位点定向突变发生,以在特定的蛋白质位置引入氨基酸.
- 培养工程化大肠杆菌以表达蛋白质.
- 双电子电子共振 (DEER) 测量用于距离测定.
- 在细胞内选择性检测自旋标记蛋白质.
主要成果:
- 成功的基因编码和稳定表达的旋转标记的氨基酸在大肠杆菌.
- 证明能够在蛋白质中的多个用户定义的部位引入氨基酸.
- 在长期表达过程中,标记的蛋白质的稳定性得到证实.
- 利用双电子电子共振 (DEER) 来测量分子内距离.
- 在完整的大肠杆菌细胞中实现了对自旋标记蛋白的选择性检测.
结论:
- 转子标记氨基酸的遗传编码为生产标记蛋白质提供了一种简化方法.
- 这种方法绕过了传统化学标签技术的局限性.
- 开发的系统为细胞内内源性蛋白质的EPR光谱学开辟了新的途径,为其本土环境中的生物过程提供了洞察力.
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