Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn(2).
Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn(2).
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定量血清和细胞内游离锌的比率生物发光锌传感器蛋白（2）。
DOI：
10.1021/acschembio.2c00227
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发表时间：
2022-06-17
影响因子：
4
通讯作者：
Merkx, Maarten
中科院分区：
文献类型：
--
作者：
Michielsen, Claire M. S.;van Aalen, Eva A.;Merkx, Maarten
锌离子荧光传感器在锌生物研究中发挥着重要作用，但其在复杂介质中的应用受到自发荧光和光散射的阻碍。生物发光传感器蛋白为这些应用提供了一种诱人的替代荧光传感器的选择，但到目前为止开发的唯一生物发光传感器蛋白BLZinCH具有有限的传感器响应和次佳的锌亲和力。在这项工作中，我们通过开发两个新的传感器家族来扩展生物发光锌离子传感器的工具箱，这两个传感器家族的发射比发生了很大的变化，并涵盖了一系列与生理相关的锌离子亲和力。Luzi平台依赖于分离的NanoLuc荧光素酶的竞争性互补，并显示出从红到蓝的强劲的2倍变化，允许在下午2点到1 nm之间定量游离锌离子。第二个平台是通过将原始BLZinCH传感器中的长而柔性的GGS接头替换为刚性多聚脯氨酸接头而开发的，从而产生了一系列BLZinCH-Pro传感器，其比率响应和生理上相关的锌离子亲和力在0.5至1 nm之间提高了3-4倍。Luzi和BLZinCH-Pro传感器都允许直接测定血清中低NM浓度的游离锌离子，为测量血清锌水平提供了一个有吸引力的替代更费力的和/或间接的方法。此外，BLZinCH-Pro传感器的遗传编码允许它们用作细胞内传感器，40-50%的传感器占有率使其非常适合在基于平板阅读器的简单测量中监测细胞内游离锌离子浓度的增加和减少，而不需要荧光显微镜。
Fluorescent Zn2+ sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn2+ affinity. In this work, we expanded the toolbox of bioluminescent Zn2+ sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn2+ affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn2+ between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3–4-fold improved ratiometric response and physiologically relevant Zn2+ affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn2+ in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40–50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn2+ concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.
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影响因子：
4.2
作者：
CHILVERS, DC;DAWSON, JB;HODGKINSON, A
通讯作者：
HODGKINSON, A
影响因子：
9.3
作者：
Maret, Wolfgang
通讯作者：
Maret, Wolfgang
DOI：
10.1016/j.jtemb.2012.04.004
发表时间：
2012-01-01
影响因子：
3.5
作者：
Prasad, Ananda S.
通讯作者：
Prasad, Ananda S.
影响因子：
3
作者：
Fukada, Toshiyuki;Yamasaki, Satoru;Nishida, Keigo;Murakami, Masaaki;Hirano, Toshio
通讯作者：
Hirano, Toshio
影响因子：
5.6
作者：
Maret W
通讯作者：
Maret W