European Synchrotron Radiation Facility, 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France.
J Am Chem Soc. 2009 Dec 23;131(50):18063-5. doi: 10.1021/ja907296v.
We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of pi conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.
我们已经在近原子分辨率下观察到光激活荧光蛋白 IrisFP 的瞬态暗状态。这种暗状态被分配给一种自由基，它要么弛豫到基态，要么演变成永久性漂白的生色团。我们利用 X 射线使自由基饱和，自由基可能是通过三重态的电子转移反应在可见光照射下形成的。X 射线衍射与晶体紫外可见吸收、荧光和拉曼数据的结合表明，IrisFP 中自由基的形成涉及到生色团的明显但可逆的变形，表明 π 共轭的暂时丧失。这些结果表明，生色团的亚甲基桥是荧光蛋白的阿喀琉斯之踵，有助于揭示 FP 中闪烁和光漂白的机制，这对于光稳定变体的合理设计具有重要意义。
