Beaumont Edward, Lambry Jean-Christophe, Robin Anne-Claire, Martasek Pavel, Blanchard-Desce Mireille, Slama-Schwok Anny
Unité 696, INSERM, Laboratory for Optics & Biosciences, UMR CNRS 7645, Ecole Polytechnique, Palaiseau, France.
Chemphyschem. 2008 Nov 10;9(16):2325-31. doi: 10.1002/cphc.200800411.
We have recently designed a nanotrigger (NT), a photoactive molecule addressing the NADPH sites of proteins. This nanotrigger has a 10(3) times larger two-photon cross-section compared to the ubiquitous NADPH cofactor. In this work, we tested whether two-photon excitation of the bound NT to NADPH sites may be used to initiate enzymatic catalysis by appropriate electron injection. To establish proof of principle, we monitored the ultrafast absorption of NT bound to the fully active endothelial NO-Synthase (eNOS) following excitation by one and two-photons at 405 and 810 nm, respectively. Electron injection from NT* to FAD in eNOS initiated the catalytic cycle in 15+/-3 ps at both exciting wavelengths. The data proved for the first time that electron transfer can be promoted by two-photon excitation. We also show that the nanotrigger decays faster in homogeneous solvents than in the NADPH site of proteins, suggesting that hindered environments modified the natural decay of NT. The nanotrigger provides a convenient way of synchronizing an ensemble of proteins in solution with a femtosecond laser pulse. The ability of NT to initiate NOS catalysis by two-photon excitation may be exploited for controlled and localized release of free NO in cells with enhanced spatial and temporal resolution.
我们最近设计了一种纳米触发器（NT），一种可作用于蛋白质NADPH位点的光活性分子。与普遍存在的NADPH辅因子相比，这种纳米触发器的双光子截面大10³倍。在这项工作中，我们测试了与NADPH位点结合的NT的双光子激发是否可用于通过适当的电子注入启动酶催化作用。为了建立原理证明，我们分别监测了在405和810 nm处单光子和双光子激发后与完全活性的内皮型一氧化氮合酶（eNOS）结合的NT的超快吸收。在两个激发波长下，从NT*到eNOS中FAD的电子注入均在15±3 ps内启动了催化循环。数据首次证明双光子激发可促进电子转移。我们还表明，纳米触发器在均匀溶剂中的衰减比在蛋白质的NADPH位点中更快，这表明受阻环境改变了NT的自然衰减。纳米触发器提供了一种用飞秒激光脉冲同步溶液中蛋白质集合的便捷方法。NT通过双光子激发启动NOS催化的能力可用于在细胞中以增强的空间和时间分辨率控制和局部释放游离NO。
