Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed
Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed
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DOI：
10.1074/jbc.m109.054973
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发表时间：
2009-11-13
影响因子：
4.8
通讯作者：
Pletnev, Vladimir
中科院分区：
文献类型：
--
作者：
Pletnev, Sergei;Gurskaya, Nadya G.;Pletnev, Vladimir
KillerRed是唯一已知的表现出显著光毒性的荧光蛋白，超过其他绿色和红色荧光蛋白至少1,000倍。KillerRed可以作为一种工具，在光动力疗法中抑制靶蛋白或杀死细胞群。然而，KillerRed光毒性的性质仍然不清楚，阻碍了更多光毒性变体的开发。在这里，我们提出了一个高分辨率的晶体学研究的结果KillerRed在活性荧光和光漂白非荧光状态。该结构的一个独特而显著的特征是从β-桶的端帽到达发色团区域的充满水的通道，这可能是负责光毒性的关键结构特征之一。基于结构的定点突变支持的KillerRed结构-功能关系的研究也揭示了最可能导致光毒性效应的关键残基。特别地，位于发色团附近的Glu（68）和Ser（119）已被指定为反应链的主要触发剂。
KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.