Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins.
Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins.
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DOI：
10.1021/acs.biochem.1c00705
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发表时间：
2022-04-05
期刊：
影响因子：
2.9
通讯作者：
Zastrow, Melissa L.
中科院分区：
文献类型：
--
作者：
Zhao, Haowen;Zastrow, Melissa L.
过渡金属如锌和铜在许多生命过程中都是必不可少的，这些金属的缺乏和毒性过载都与各种疾病有关。金属荧光传感器是研究金属离子在生物系统生理和病理中作用的有力工具。绿色荧光蛋白(GFP)及其衍生物在基于蛋白质的传感器设计中得到了高度的利用，但由于这些蛋白质需要氧气才能变成荧光，因此在厌氧系统中的应用受到限制。基于细菌藻色素的单体近红外荧光蛋白(MiRFPs)共价结合一个Blin辅因子，可以外源添加到厌氧细胞中。MiRFP的发射波长也可以延伸到700 nm，这对成像应用很有价值。在这里，我们评估了miRFP670和miRFP709作为单一荧光蛋白金属离子传感器平台的适用性。我们发现，二价金属离子，如锌、钴、镍和铜，可以猝灭纯miRFPs荧光强度的~6-20%(Zn2+、Co2+和Ni2+)和近90%(Cu2+)，并在表达miRFPs的活大肠杆菌细胞中产生类似的影响。6×组氨酸标签的存在影响金属猝灭，但在没有His6标签的情况下，Cu2+诱导的猝灭和皮摩尔结合亲和力在试管和活细菌细胞中都保持不变。通过比较Cu2+和Cu2+对miRFP670和miRFP709的猝灭结果，结合吸收光谱和已报道的晶体结构，我们提出了胆绿素IXα发色团附近的表面金属结合部位。
Transition metals such as zinc and copper are essential in numerous life processes, and both deficiency and toxic overload of these metals are associated with various diseases. Fluorescent metal sensors are powerful tools for studying the roles of metal ions in the physiology and pathology of biological systems. Green fluorescent protein (GFP) and its derivatives are highly utilized for protein-based sensor design, but application to anaerobic systems is limited because these proteins require oxygen to become fluorescent. Bacteriophytochrome-based monomeric near-infrared fluorescent proteins (miRFPs) covalently bind a bilin cofactor, which can be added exogenously for anaerobic cells. miRFPs can also have emission wavelengths extending to >700 nm, which is valuable for imaging applications. Here, we evaluated the suitability of miRFP670 and miRFP709 as platforms for single fluorescent protein metal ion sensors. We found that divalent metal ions like Zn2+, Co2+, Ni2+, and Cu2+ can quench from ~6–20% (Zn2+, Co2+, and Ni2+) and up to nearly 90% (Cu2+) of the fluorescence intensity of pure miRFPs and have similar impacts in live Escherichia coli cells expressing miRFPs. The presence of a 6× histidine tag for purification influences metal quenching, but significant Cu2+-induced quenching and a picomolar binding affinity are retained in the absence of the His6 tag in both cuvettes and live bacterial cells. By comparing the Cu2+ and Cu+-induced quenching results for miRFP670 and miRFP709 and through examining absorption spectra and previously reported crystal structures, we propose a surface metal binding site near the biliverdin IXα chromophore.
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