A genetically encoded fluorescent sensor for manganese(II), engineered from lanmodulin.
A genetically encoded fluorescent sensor for manganese(II), engineered from lanmodulin.
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DOI：
10.1073/pnas.2212723119
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发表时间：
2022-12-20
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
--
作者：
实际上，不存在用于细胞中锰 (II) 实时成像的化学生物学工具。这些工具可以帮助了解锰在氧化防御、光合作用和其他重要生物过程中如何发挥作用，无论是作为酶辅助因子与蛋白质结合还是在不稳定的锰库中未结合。在这里，我们展示了最近发现的天然镧系元素结合蛋白可以被重新设计，以对细胞中最重要的干扰金属（镁、铁和钙）具有强选择性地响应锰。这种基因编码的荧光传感器实时报告细菌细胞中的锰通量，为研究锰生理学的新方法奠定了基础。更广泛地说，它提出了将非天然金属选择性重新设计成蛋白质以实现广泛应用的一般策略。选择性金属结合位点的设计在小分子和大分子化学中都是一个挑战。选择性识别锰 (II)（欧文-威廉姆斯系列所描述的第一行过渡金属离子，往往以最低的亲和力与配体结合）特别困难。因此，缺乏用于研究活细胞中锰生理学的化学生物学工具，而这将促进对光合作用、宿主与病原体相互作用和神经生物学的理解。在这里，我们报告了将镧系元素结合蛋白 lanmodulin 合理重新设计为 MnII、MnLaMP1 和 MnLaMP2 的基因编码荧光传感器。这些传感器的有效 Kd(MnII) 分别为 29 和 7 µM，无法在体外和体内选择性检测 MnII。我们应用这两种传感器来可视化细菌不稳定锰池的动力学。生物物理学研究表明协调的溶剂和疏水相互作用在传感器的选择性中的重要性。我们的结果确立了兰莫杜林作为一种多功能支架，用于设计基于蛋白质的选择性生物传感器和 f 区以外的金属螯合剂。
Virtually no chemical biology tools exist for real-time imaging of manganese(II) in cells. Such tools could help to understand how manganese functions in oxidative defense, photosynthesis, and other important biological processes, both when protein-bound as an enzyme cofactor and unbound in the labile manganese pool. Here we show that a recently discovered native lanthanide-binding protein can be re-engineered to respond to manganese with strong selectivity over the most important interfering metals in cells (magnesium, iron, and calcium). This genetically encoded fluorescent sensor reports manganese fluxes in bacterial cells in real time, laying the foundation for a new approach to studying manganese physiology. More broadly, it suggests general strategies for re-engineering non-native metal selectivity into proteins for wide-ranging applications. The design of selective metal-binding sites is a challenge in both small-molecule and macromolecular chemistry. Selective recognition of manganese (II)—the first-row transition metal ion that tends to bind with the lowest affinity to ligands, as described by the Irving-Williams series—is particularly difficult. As a result, there is a dearth of chemical biology tools with which to study manganese physiology in live cells, which would advance understanding of photosynthesis, host-pathogen interactions, and neurobiology. Here we report the rational re-engineering of the lanthanide-binding protein, lanmodulin, into genetically encoded fluorescent sensors for MnII, MnLaMP1 and MnLaMP2. These sensors with effective Kd(MnII) of 29 and 7 µM, respectively, defy the Irving-Williams series to selectively detect MnII in vitro and in vivo. We apply both sensors to visualize kinetics of bacterial labile manganese pools. Biophysical studies indicate the importance of coordinated solvent and hydrophobic interactions in the sensors’ selectivity. Our results establish lanmodulin as a versatile scaffold for design of selective protein-based biosensors and chelators for metals beyond the f-block.
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影响因子：
3.6
作者：
Anjem A;Varghese S;Imlay JA
通讯作者：
Imlay JA
影响因子：
7.8
作者：
Foster AW;Young TR;Chivers PT;Robinson NJ
通讯作者：
Robinson NJ
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
27.5
作者：
Eisenhut, Marion;Hoecker, Natalie;Schneider, Anja
通讯作者：
Schneider, Anja
DOI：
10.1085/jgp.49.2.221
发表时间：
1965-11-01
期刊：
The Journal of general physiology
影响因子：
--
作者：
Epstein W;Schultz SG
通讯作者：
Schultz SG