Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.
Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.
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DOI：
10.1016/j.chembiol.2017.02.008
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发表时间：
2017-03-16
影响因子：
8.6
通讯作者：
Arnold FH
中科院分区：
文献类型：
--
作者：
Herwig L;Rice AJ;Bedbrook CN;Zhang RK;Lignell A;Cahn JKB;Renata H;Dodani SC;Cho I;Cai L;Gradinaru V;Arnold FH
通过设计微生物视紫红质 Archaerhodopsin-3 (Arch) 来结合合成生色团、部花青视黄醛，代替天然生色团全反式视黄醛 (ATR)，我们生成了一种具有异常明亮和前所未有的红移近红外 (NIR) 荧光的蛋白质。我们发现，发色团取代产生了荧光 Arch 复合物，相对于 ATR 结合的野生型 Arch 而言，其红红激发位移为 200 nm，最大发射波长为 772 nm。该复合物的定向进化产生了具有 pH 敏感 NIR 荧光的变体，其分子亮度比最亮的 ATR 结合的 Arch 变体高 8.5 倍。所得蛋白质非常适合细菌成像；表达和稳定性尚未针对哺乳动物细胞成像进行优化。通过靶向蛋白质及其发色团，我们克服了将明亮的近红外荧光工程转化为古视紫红质相关的固有挑战。这项工作展示了一种有效的策略，可以将非天然的、定制的特性转化为微生物视蛋白，以及与成像和询问生物系统相关的特性。 Herwig 等人使用发色团替换和定向进化的组合方法。工程化的荧光古视紫红质变体具有前所未有的近红外（NIR）激发和发射。进化后的变体表现出 pH 敏感性、增强的荧光分子亮度和改进的合成发色团亲和力。
By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch-complex with a 200 nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems. Using a combined approach of chromophore substitution and directed evolution, Herwig et al. engineered fluorescent Archaerhodopsin variants with unprecedented near-infrared (NIR) excitation and emission. Evolved variants display pH sensitivity, enhanced fluorescent molecular brightness and improved synthetic chromophore affinity.