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首页> 外文期刊>Chemistry: A European journal >Selective two-step labeling of proteins with an off/on fluorescent probe
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Selective two-step labeling of proteins with an off/on fluorescent probe

机译：使用关闭/打开的荧光探针对蛋白质进行选择性的两步标记

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摘要

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni ~(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni ~(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni ~(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis _6-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His _6Cys-tagged enhanced blue fluorescent protein (EBFP), but not His _6-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.
机译：我们提出了一种适用于蛋白质的选择性两步标记的关闭/开启荧光探针的新颖设计策略。为了验证该策略，我们设计并合成了以半胱氨酸修饰的六组氨酸（His）标签为靶的荧光探针1-Ni〜（2+）。探针由与次氮基三乙酸（NTA）-Ni〜（2+）共轭的二氯荧光素（作为His-tag识别位点）和2,4-二硝基苯基醚部分组成，该部分可通过光致电子转移（PeT）来淬灭探针的荧光与2,4-二硝基苯醚（供体激发的PeT; d-PeT）激发荧光团，并且与半胱氨酸具有反应性。通过NTA-Ni〜（2+）部分识别组氨酸标签，然后通过与修饰标签的半胱氨酸残基进行邻近增强反应，去除2,4-二硝基苯基醚淬灭剂;这导致荧光显着增加。将带有半胱氨酸残基的His-tag肽添加到探针水溶液中可在10分钟内增加约20倍的荧光增量，这是迄今为止基于可见光激发的基于His的肽标签的荧光探针获得的最大荧光增强。此外，我们无需任何洗涤步骤即可成功地将拴在微珠上的CysHis -6肽可视化。该探针还显示存在His _6Cys标签的增强型蓝色荧光蛋白（EBFP），但没有His _6标签的EBFP时，荧光增量较大。我们认为该系统优于可干扰蛋白质折叠，运输和功能的大型荧光标签（例如，绿色荧光蛋白：27 kDa），也优于现有的小型标签，后者通常在目标识别后显示很少的荧光增加，因此需要冲洗步骤。此策略也应适用于其他标签。

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