用DEER/PELDOR观察到大肠杆菌细胞中膜载体的联结体诱导的形状变化
Benesh Joseph1, Arthur Sikora2, David S Cafiso2
1Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, University of Frankfurt , Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany.
Journal of the American Chemical Society
|January 23, 2016
概括
研究人员使用脉冲电子双共振 (PELDOR) 来观察完整细胞中的膜蛋白形状变化. 这种方法绕过了传统的净化步骤,使细胞内结构研究成为可能.
科学领域:
- 结构生物学
- 生物物理
- 膜蛋白研究
背景情况:
- 在细胞环境中确定膜蛋白的高分辨率结构和构造变化仍然是一个挑战.
- 脉冲电子-电子双共振 (PELDOR) 是有效的监测纯化膜蛋白复合体的结构变化.
- 目前的方法往往需要清洗剂的提取,净化和复制,这可能会改变蛋白质的结构和功能.
研究的目的:
- 证明使用PELDOR观察完整细胞内膜蛋白的结构变化的可行性.
- 在它们的原生细胞环境中开发一种特定的外膜蛋白标签策略.
- 鉴定胺转运体BtuB的联体诱导的形状变化.
主要方法:
- 在完整的大肠杆菌细胞上使用脉冲电子双共振 (PELDOR).
- 开发了一种选择性标记策略,利用外膜蛋白中缺乏反应性囊和选择性减少周等离子体旋转标签.
- 专门标记了可巴胺转运体BtuB,以监测其第二个细胞外循环的结构变化.
主要成果:
- 在完整的细菌细胞内观察膜蛋白 (BtuB) 的构造变化.
- 在BtuB的第二个细胞外环中表现出联体结合诱导的形状变化.
- PELDOR数据与高分辨率的晶体结构一致,验证了细胞内方法.
结论:
- 这项研究为细胞内膜蛋白的PELDOR研究提供了第一个概念验证.
- 开发的方法避免了洗剂处理和净化,保护了原生细胞环境.
- 这种方法为研究外膜蛋白的结构,功能和相互作用开辟了新的途径.
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