Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP
Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP
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DOI：
10.1016/j.jmb.2019.06.005
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发表时间：
2019-08-09
影响因子：
5.6
通讯作者：
Kim, Jeong-Sun
中科院分区：
文献类型：
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作者：
Seo, Pil-Won;Jo, Eun-Seo;Kim, Jeong-Sun
荧光蛋白，如绿色荧光蛋白，用于检测细胞成分和事件。然而，绿色荧光蛋白及其衍生物在厌氧条件下的用途有限，需要较长的成熟时间。另一方面，依赖NADPH的蓝色荧光蛋白(BFP)不需要对残基进行氧化修饰，在好氧和厌氧系统中都能立即发挥作用。BFP蛋白属于短链脱氢酶/还原酶(SDR)蛋白家族，在底物存在下，其荧光性质随反应时间而变化。为了开发一种更好的不依赖于氧化还原状态的荧光报告分子，我们从含有和不含有NADPH的土壤中分离得到了mBFP四聚体的晶体结构。除了已知的区域外，结构导向突变研究已经确定了几个有助于mBFP荧光的残基，包括结合的NADPH的烟酰胺部分附近的两个芳香族残基(F97和Y157)。即使在底物存在的情况下，Y157(Y157H)上的单个组氨酸突变也可以产生更稳定的、与时间无关的荧光。此外，我们还发现了另一种SDR蛋白，它也可以发出蓝色荧光。这些结果为BFP作为一种稳定的细胞报告蛋白的广泛应用开辟了新的可能性，而不受亚细胞环境的影响。(C)2019爱思唯尔有限公司。保留所有权利。
Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments. (C) 2019 Elsevier Ltd. All rights reserved.