Department of Chemistry, Zukunftskolleg, and Konstanz Research School Chemical Biology, University of Konstanz , Universitätsstraße 10, 78457 Konstanz, Germany.
J Am Chem Soc. 2014 Jan 29;136(4):1238-41. doi: 10.1021/ja411535q. Epub 2014 Jan 15.
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.
我们报告了一种非典型的、自旋标记氨基酸在大肠杆菌中的遗传编码。这使得在细胞内生物合成自旋标记的蛋白质成为可能，并避免了通常用于蛋白质电子顺磁共振（EPR）研究的任何化学标记步骤。这种氨基酸可以在蛋白质的多个用户定义的位置引入，并且即使在长时间表达的情况下在大肠杆菌中也很稳定。它可以通过双电子电子共振测量报告蛋白质中的分子内距离分布。此外，自旋标记蛋白的信号可以在细胞中选择性地检测到。这为内源性蛋白质的细胞内 EPR 研究提供了新的思路。
