Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids
Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids
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DOI：
10.1021/jacs.3c00967
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发表时间：
2023-06-26
影响因子：
15
通讯作者：
Mehl, Ryan A.
中科院分区：
文献类型：
--
作者：
Jana, Subhashis;Evans, Eric G. B.;Mehl, Ryan A.
定点自旋标记（SDSL）技术与双电子-电子共振（DEER）技术相结合，已成为测定生物大分子结构状态和构象平衡的有力技术。DEER结合原位SDSL在活细胞中是具有挑战性的，因为目前的生物正交标记方法太慢，无法在细胞减少信号丢失之前用低浓度的自旋标记物进行完全标记。在这里，我们通过在大肠杆菌和人HEK 293 T细胞中表达的蛋白质的多个位点上遗传编码一个新的小的、四嗪携带的非经典氨基酸（Tet-v4.0）家族来克服这种限制。我们通过开发一系列应变反式环辛烯（sTCO）功能化的氮氧自由基，实现了对含Tet-v4.0蛋白质的特异性和定量自旋标记，其中包括一种在细胞中具有增强稳定性的偕二乙基取代氮氧自由基，其速率常数可超过10（6）M-1 s（-1）。Tet-v4.0/sTCO反应的可调节速度允许在几分钟内有效地自旋标记活细胞中的蛋白质，仅需要亚微摩尔浓度的sTCO-氮氧化物。从完整细胞记录的DEER显示距离分布与从体外纯化和标记的蛋白质测量的距离分布非常一致。此外，DEER还能在体外解析Tet-v4.0掺入和自旋标记的MBP的麦芽糖依赖性构象变化，并支持在HEK 293 T细胞中MBP突变体的构象状态归属。我们预计该系统的异常反应速率，结合所得自旋标记物的相对较短和刚性的侧链，将使蛋白质的结构/功能研究直接在细胞中进行，而不需要任何蛋白质纯化。
Site-directedspin-labeling (SDSL)in combinationwith doubleelectron-electron resonance (DEER) spectroscopy hasemerged as a powerful technique for determining both the structuralstates and the conformational equilibria of biomacromolecules. DEERcombined with in situ SDSL in live cells is challengingsince current bioorthogonal labeling approaches are too slow to allowfor complete labeling with low concentrations of spin label priorto loss of signal from cellular reduction. Here, we overcome thislimitation by genetically encoding a novel family of small, tetrazine-bearingnoncanonical amino acids (Tet-v4.0) at multiple sites in proteinsexpressed in Escherichia coli and inhuman HEK293T cells. We achieved specific and quantitative spin-labelingof Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides includinga gem-diethyl-substituted nitroxide with enhancedstability in cells with rate constants that can exceed 10(6) M-1 s(-1). The remarkablespeed of the Tet-v4.0/sTCO reaction allowed efficient spin-labelingof proteins in live cells within minutes, requiring only sub-micromolarconcentrations of sTCO-nitroxide. DEER recorded from intact cellsrevealed distance distributions in good agreement with those measuredfrom proteins purified and labeled in vitro. Furthermore,DEER was able to resolve the maltose-dependent conformational changeof Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutantwithin HEK293T cells. We anticipate the exceptional reaction ratesof this system, combined with the relatively short and rigid sidechains of the resulting spin labels, will enable structure/functionstudies of proteins directly in cells, without any requirements forprotein purification.