Pletneva Nadya V, Pletnev Vladimir Z, Sarkisyan Karen S, Gorbachev Dmitry A, Egorov Evgeny S, Mishin Alexander S, Lukyanov Konstantin A, Dauter Zbigniew, Pletnev Sergei
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
Faculty of Biology, Moscow State University, Moscow, Russian Federation.
PLoS One. 2015 Dec 23;10(12):e0145740. doi: 10.1371/journal.pone.0145740. eCollection 2015.
Phototoxic fluorescent proteins represent a sparse group of genetically encoded photosensitizers that could be used for precise light-induced inactivation of target proteins, DNA damage, and cell killing. Only two such GFP-based fluorescent proteins (FPs), KillerRed and its monomeric variant SuperNova, were described up to date. Here, we present a crystallographic study of their two orange successors, dimeric KillerOrange and monomeric mKillerOrange, at 1.81 and 1.57 Å resolution, respectively. They are the first orange-emitting protein photosensitizers with a tryptophan-based chromophore (Gln65-Trp66-Gly67). Same as their red progenitors, both orange photosensitizers have a water-filled channel connecting the chromophore to the β-barrel exterior and enabling transport of ROS. In both proteins, Trp66 of the chromophore adopts an unusual trans-cis conformation stabilized by H-bond with the nearby Gln159. This trans-cis conformation along with the water channel was shown to be a key structural feature providing bright orange emission and phototoxicity of both examined orange photosensitizers.
光毒性荧光蛋白是一组数量稀少的基因编码光敏剂，可用于精确光诱导靶蛋白失活、DNA损伤和细胞杀伤。截至目前，仅描述了两种基于绿色荧光蛋白（GFP）的荧光蛋白（FPs），即KillerRed及其单体变体SuperNova。在此，我们分别以1.81 Å和1.57 Å的分辨率对它们的两种橙色后继者，二聚体KillerOrange和单体mKillerOrange进行了晶体学研究。它们是首批以基于色氨酸的发色团（Gln65-Trp66-Gly67）发出橙色荧光的蛋白光敏剂。与它们的红色前身一样，这两种橙色光敏剂都有一个充满水的通道，将发色团连接到β桶外部，并能够运输活性氧。在这两种蛋白质中，发色团的Trp66都采用了一种不寻常的反式-顺式构象，通过与附近的Gln159形成氢键而稳定。这种反式-顺式构象以及水通道被证明是提供两种橙色光敏剂明亮橙色发射和光毒性的关键结构特征。
