Carpentier Philippe, Violot Sebastien, Blanchoin Laurent, Bourgeois Dominique
Laboratoire de Cristallographie et Cristallogenèse des Protéines, IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, Université Joseph Fourier, Grenoble, France.
FEBS Lett. 2009 Sep 3;583(17):2839-42. doi: 10.1016/j.febslet.2009.07.041. Epub 2009 Jul 30.
The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by "pre-bleached" KillerRed.
红色荧光蛋白KillerRed由水螅虫纲的发色蛋白anm2CP改造而来，据报道它可通过发色团辅助光灭活（CALI）效应诱导强烈的细胞毒性。在此，我们展示了KillerRed处于天然状态和漂白状态下的X射线结构。揭示了一条长长的充满水的通道，该通道将发色团的亚甲基桥与溶剂相连。此通道有助于氧气以及与激发态发色团反应形成的活性氧（ROS）的传输。讨论了用于生产KillerRed的关键突变的功能作用，揭示了漂白状态下强烈的发色团畸变，并提出了ROS产生和自我保护的机制。表征了一种部分成熟、抗光、发绿色荧光的状态的存在，这解释了“预漂白”的KillerRed增强的CALI效应。
