The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

中文翻译：

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利用蛋白质设计和定向进化来单体化明亮的近红外荧光蛋白质

小红外荧光蛋白 (smURFP) 是一种明亮的近红外 (NIR) 荧光蛋白 (FP)，可形成二聚体并在其二聚体界面结合其荧光发色团胆绿素。为了设计基于 smURFP 的单体 NIR FP 可能更适合生物成像，我们采用蛋白质设计，用两个螺旋的新片段延伸蛋白质主链，屏蔽原始二聚体界面，同时覆盖胆绿素结合袋代替第二条链。原来的二聚体。我们通过实验对 13 种设计进行了表征，并获得了具有弱荧光的单体蛋白。我们通过两轮定向进化增强了这种设计的蛋白质的荧光，并获得了设计的单体 smURFP (DMsmURFP)，这是一种明亮、稳定的单体近红外 FP，分子量为 19.6 kDa。我们确定了 DMsmURFP 的 apo 状态和与胆绿素复合物的晶体结构，这证实了设计的结构。 DMsmURFP 在哺乳动物系统体内成像中的应用得到了证实。这里使用的基于主链设计的策略也可以应用于单体化具有亚基间功能位点的其他天然多聚蛋白。