Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Bioorg Med Chem. 2011 Feb 1;19(3):1079-84. doi: 10.1016/j.bmc.2010.07.014. Epub 2010 Aug 5.
Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.
过氧化氢是一种重要的第二信使，通过选择性氧化蛋白质中具有还原活性的硫醇来控制细胞内信号级联反应。可以使用 HyPer（一种比率型遗传编码荧光探针）实时跟踪细胞内 [H2O2] 的变化。尽管由于其传感结构域源自大肠杆菌 OxyR 蛋白的特性，HyPer 对 H2O2 具有敏感性和选择性，但许多应用可能受益于指示剂动态范围的改善。我们在此报告 HyPer-2，这是一种满足这一需求的探针。在达到 [H2O2] 暴露的饱和度后，HyPer-2 的 F500/F420 比值增加了六倍，而 HyPer 增加了三倍。HyPer-2 是通过 HyPer 中的单点突变 A406V 产生的，该突变对应于 wtOxyR 中的 A233V。该突变先前被证明会破坏 OxyR 二聚体中单体之间的界面。然而，在 HyPer-2 中，A233V 突变稳定了二聚体并扩展了探针的动态范围。
