Karim Christine B, Zhang Zhiwen, Thomas David D
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Nat Protoc. 2007;2(1):42-9. doi: 10.1038/nprot.2007.2.
A procedure is described for the synthetic incorporation into membrane proteins of the non-natural amino acid TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid), which is coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by electron paramagnetic resonance (EPR). Also included is a protocol for the functional reconstitution of the spin-labeled protein in lipid vesicles. This protocol can be completed in 17 d.
本文描述了一种将非天然氨基酸TOAC（2,2,6,6-四甲基哌啶-1-氧基-4-氨基-4-羧酸）合成掺入膜蛋白的方法，该氨基酸与α-碳刚性偶联，可通过电子顺磁共振（EPR）直接检测肽主链动力学。文中还包括了在脂质囊泡中对自旋标记蛋白进行功能重建的方案。该方案可在17天内完成。
