Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan.
Department of Physiological Sciences, The Graduate University for Advanced Studies, Hayama, Kanagawa, 240-0193, Japan.
Sci Rep. 2019 Aug 19;9(1):12072. doi: 10.1038/s41598-019-48604-4.
Here we developed an orange light-absorbing chromoprotein named ShadowR as a novel acceptor for performing fluorescence lifetime imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) measurement in living cells. ShadowR was generated by replacing hydrophobic amino acids located at the surface of the chromoprotein Ultramarine with hydrophilic amino acids in order to reduce non-specific interactions with cytosolic proteins. Similar to Ultramarine, ShadowR shows high absorption capacity and no fluorescence. However, it exhibits reduced non-specific binding to cytosolic proteins and is highly expressed in HeLa cells. Using tandem constructs and a LOVTRAP system, we showed that ShadowR can be used as a FRET acceptor in combination with donor mRuby2 or mScarlet in HeLa cells. Thus, ShadowR is a useful, novel FLIM-FRET acceptor.
在这里，我们开发了一种橙色吸光的色蛋白，名为 ShadowR，作为一种新型的受体，用于在活细胞中进行荧光寿命成像显微镜的Förster 共振能量转移（FLIM-FRET）测量。ShadowR 通过用亲水氨基酸取代位于色蛋白 Ultramarine 表面的疏水性氨基酸来产生，以减少与细胞质蛋白的非特异性相互作用。与 Ultramarine 类似，ShadowR 具有高的吸收能力和无荧光。然而，它表现出与细胞质蛋白的非特异性结合减少，并且在 HeLa 细胞中高度表达。使用串联构建体和 LOVTRAP 系统，我们表明 ShadowR 可以与供体 mRuby2 或 mScarlet 结合，作为 HeLa 细胞中 FRET 受体使用。因此，ShadowR 是一种有用的新型 FLIM-FRET 受体。
