Department of Chemistry, University of Houston, Houston, Texas 77204, United States.
ACS Sens. 2022 Nov 25;7(11):3369-3378. doi: 10.1021/acssensors.2c01376. Epub 2022 Oct 25.
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 (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 (CreiLOV) significantly altered metal affinity and selectivity, yielding a zinc sensor. The fluorescence intensity and lifetime of CreiLOV were reversibly quenched by Zn ions with a biologically relevant affinity (apparent dissociation constant, , of 1 nM). Copper quenching of CreiLOV was retained but with several orders of magnitude higher affinity than CreiLOV ( = 0.066 fM for Cu, 5.4 fM for Cu) and partial reversibility. We also show that CreiLOV is an excellent intensity- and lifetime-based zinc sensor in aerobic and anaerobic live bacterial cells. Zn-induced fluorescence quenching is reversible over several cycles in cell suspensions and can be imaged by fluorescence microscopy. CreiLOV 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.
黄素基荧光蛋白（FbFPs）是一类荧光报告蛋白，它们可以在无氧条件下进行荧光团的掺入，这是相对于绿色荧光蛋白（GFPs）和 mFruits 的一个重要优势。一种来源于 （CreiLOV）的 FbFP 是设计新型金属传感器的有前途的平台。一些 FbFPs 本身被金属离子猝灭，但金属结合的位置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 的定点突变来探测导致荧光猝灭的假设铜（II）结合位点。大多数突变改变了荧光猝灭水平，支持了提出的位点。一个关键的突变是用半胱氨酸取代天冬酰胺（CreiLOV），这显著改变了金属亲和力和选择性，产生了锌传感器。CreiLOV 的荧光强度和寿命可被 Zn 离子可逆猝灭，具有生物相关的亲和力（表观解离常数 ， 为 1 nM）。CreiLOV 对铜的猝灭仍然存在，但亲和力比 CreiLOV 高几个数量级（铜的 = 0.066 fM，铜的 = 5.4 fM），且部分可逆。我们还表明，CreiLOV 是一种在有氧和无氧活细菌细胞中基于强度和寿命的出色锌传感器。在细胞悬浮液中，Zn 诱导的荧光猝灭可在几个循环中可逆，并可通过荧光显微镜成像。CreiLOV 是一种新型的无氧依赖性金属传感器，它极大地扩展了当前基于荧光蛋白的金属传感器工具包，并将允许对以前由于使用氧依赖性 GFP 而无法研究的厌氧和低氧系统进行研究。
