Mutant Flavin-Based Fluorescent Protein Sensors for Detecting Intracellular Zinc and Copper in Escherichia coli.
Mutant Flavin-Based Fluorescent Protein Sensors for Detecting Intracellular Zinc and Copper in Escherichia coli.
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DOI：
10.1021/acssensors.2c01376
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发表时间：
2022-11-25
期刊：
影响因子：
8.9
通讯作者：
Zastrow, Melissa L.
中科院分区：
文献类型：
--
作者：
Zou, Wenping;Nguyen, Hazel N.;Zastrow, Melissa L.
关键词：
基于黄素的荧光蛋白（Flavin-based fluorescent proteins，FbFP）是一类不依赖于氧的荧光团掺入的荧光报告蛋白，与绿色荧光蛋白（green fluorescent proteins，GFP）和mFruits相比具有重要的优势。来源于莱茵衣藻（CreiLOV）的FbFP是用于设计新的金属传感器的有前途的平台。一些FbFP本质上被金属离子淬灭，但金属在哪里结合以及如何调节金属亲和力的问题尚未得到解决。我们使用CreiLOV的定点诱变来探测导致荧光淬灭的假设的铜（II）结合位点。大多数突变改变了荧光猝灭水平，支持所提出的位点。一个关键的突变引入第二个半胱氨酸残基代替天冬酰胺（CreiLOVN 41 C）显着改变金属亲和力和选择性，产生锌传感器。CreiLOVN 41 C的荧光强度和寿命被具有生物学相关亲和力的Zn 2+离子可逆地猝灭（表观解离常数Kd为1 nM）。保留了CreiLOVN 41 C的铜淬灭，但具有比CreiLOV高几个数量级的亲和力（对于Cu 2+，Kd = 0.066fM，对于Cu+，Kd = 5.4fM）和部分可逆性。我们还表明，CreiLOVN 41 C是一个优秀的强度和寿命为基础的锌传感器在有氧和厌氧活细菌细胞。Zn 2+诱导的荧光淬灭在大肠杆菌细胞悬浮液中经过几个循环是可逆的，并且可以通过荧光显微镜成像。CreiLOVN 41 C是一种新型的不依赖于氧的金属传感器，它大大扩展了目前基于荧光蛋白的金属传感器工具箱，并将允许研究以前因使用氧依赖性GFP而被排除在外的厌氧和低氧系统。
Flavin-based fluorescent proteins (FbFPs) are a class of fluorescent reporters that undergo oxygen-independent fluorophore incorporation, which is an important advantage over green fluorescent proteins (GFPs) and mFruits. A FbFP derived from Chlamydomonas reinhardtii (CreiLOV) is a promising platform for designing new metal sensors. Some FbFPs are intrinsically quenched by metal ions, but the question of where metals bind and how to tune metal affinity has not been addressed. We used site-directed mutagenesis of CreiLOV to probe a hypothesized copper(II) binding site that led to fluorescence quenching. Most mutations changed the fluorescence quenching level, supporting the proposed site. One key mutation introducing a second cysteine residue in place of asparagine (CreiLOVN41C) significantly altered metal affinity and selectivity, yielding a zinc sensor. The fluorescence intensity and lifetime of CreiLOVN41C were reversibly quenched by Zn2+ ions with a biologically relevant affinity (apparent dissociation constant, Kd, of 1 nM). Copper quenching of CreiLOVN41C was retained but with several orders of magnitude higher affinity than CreiLOV (Kd = 0.066 fM for Cu2+, 5.4 fM for Cu+) and partial reversibility. We also show that CreiLOVN41C is an excellent intensity- and lifetime-based zinc sensor in aerobic and anaerobic live bacterial cells. Zn2+-induced fluorescence quenching is reversible over several cycles in Escherichia coli cell suspensions and can be imaged by fluorescence microscopy. CreiLOVN41C is a novel oxygen-independent metal sensor that significantly expands the current fluorescent protein-based toolbox of metal sensors and will allow for studies of anaerobic and low oxygen systems previously precluded by the use of oxygen-dependent GFPs.
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