A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance Measurements
A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance Measurements
复制标题
DOI：
10.1021/ja411535q
复制
发表时间：
2014-01-29
影响因子：
15
通讯作者：
Summerer, Daniel
中科院分区：
文献类型：
--
作者：
Schmidt, Moritz J.;Borbas, Julia;Summerer, Daniel
我们报告了大肠杆菌中一种非经典的自旋标记氨基酸的遗传编码。这使得自旋标记蛋白质的细胞内生物合成成为可能，并避免了蛋白质电子顺磁共振（EPR）研究通常需要的任何化学标记步骤。该氨基酸可以引入蛋白质的多个用户定义位点，并且在大肠杆菌中稳定。大肠杆菌中表达，甚至延长表达时间。它可以通过双电子电子共振测量来报告蛋白质分子内的距离分布。此外，自旋标记蛋白的信号可以在细胞中被选择性地检测到。这为内源性蛋白质的细胞内EPR研究提供了新的视角。
We report the genetic encoding of a noncanonical, spin-labeled amino acid in Escherichia coli. This enables the intracellular biosynthesis of spin-labeled proteins and obviates the need for any chemical labeling step usually required for protein electron paramagnetic resonance (EPR) studies. The amino acid can be introduced at multiple, user-defined sites of a protein and is stable in E. coli even for prolonged expression times. It can report intramolecular distance distributions in proteins by double-electron electron resonance measurements. Moreover, the signal of spin-labeled protein can be selectively detected in cells. This provides elegant new perspectives for in-cell EPR studies of endogenous proteins.