Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Mol Pharm. 2012 Apr 2;9(4):1024-9. doi: 10.1021/mp200615m. Epub 2012 Mar 13.
We show that fluorine NMR can be used to monitor the insertion and change in conformation of a ¹⁹F-labeled cell-penetrating peptide upon interacting with the cellular plasma membrane. α-Synuclein and a construct comprising a cell-penetrating peptide covalently attached to its N-terminus were studied. Important information about the interaction of the proteins with CHO-K1 cells was obtained by monitoring the diminution of ¹⁹F resonances of 3-fluoro-l-tyrosine labeled proteins. For α-synuclein, a decrease in the resonance from position 39 was observed indicating that only the N-terminal third region of the protein interacts with plasma membrane. However, when the fusion construct was incubated with the cells, a decrease in the resonance from the fusion peptide region was noted with no change in the resonances from α-synuclein region. Longer incubation, studied by using confocal fluorescence microscopy, revealed that the fusion construct translocates into the cells, but α-synuclein alone did not cross the membrane in significant amounts.
我们表明，氟 NMR 可用于监测与细胞膜相互作用时，¹⁹F 标记的穿膜肽的插入和构象变化。我们研究了α-突触核蛋白和一种包含穿膜肽共价连接到其 N 末端的构建体。通过监测 3-氟-L-酪氨酸标记的蛋白质的 ¹⁹F 共振的减少，获得了有关蛋白质与 CHO-K1 细胞相互作用的重要信息。对于α-突触核蛋白，观察到位置 39 的共振减少，表明只有蛋白质的 N 端前三分之一区域与质膜相互作用。然而，当融合构建体与细胞孵育时，融合肽区域的共振减少，而α-突触核蛋白区域的共振没有变化。通过使用共聚焦荧光显微镜研究更长时间的孵育，揭示了融合构建体转位到细胞中，但α-突触核蛋白本身没有大量穿过膜。
