Spin labels for 19F ENDOR distance determination: resolution, sensitivity and distance predictability†

IF 2.9 3区 化学 Q3 CHEMISTRY, PHYSICAL Physical Chemistry Chemical Physics Pub Date : 2024-10-11 DOI:10.1039/D4CP02996H
Alexey Bogdanov, Longfei Gao, Arina Dalaloyan, Wenkai Zhu, Manas Seal, Xun-Cheng Su, Veronica Frydman, Yangping Liu, Angela M. Gronenborn and Daniella Goldfarb
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Abstract

19F electron-nuclear double resonance (ENDOR) has emerged as an attractive method for determining distance distributions in biomolecules in the range of 0.7–2 nm, which is not easily accessible by pulsed electron dipolar spectroscopy. The 19F ENDOR approach relies on spin labeling, and in this work, we compare various labels’ performance. Four protein variants of GB1 and ubiquitin bearing fluorinated residues were labeled at the same site with nitroxide and trityl radicals and a Gd(III) chelate. Additionally, a double-histidine variant of GB1 was labeled with a Cu(II) nitrilotriacetic acid chelate. ENDOR measurements were carried out at W-band (95 GHz) where 19F signals are well separated from 1H signals. Differences in sensitivity were observed, with Gd(III) chelates providing the highest signal-to-noise ratio. The new trityl label, OXMA, devoid of methyl groups, exhibited a sufficiently long phase memory time to provide an acceptable sensitivity. However, the longer tether of this label effectively reduces the maximum accessible distance between the 19F and the Cα of the spin-labeling site. The nitroxide and Cu(II) labels provide valuable additional geometric insights via orientation selection. Prediction of electron–nuclear distances based on the known structures of the proteins were the closest to the experimental values for Gd(III) labels, and distances obtained for Cu(II) labeled GB1 are in good agreement with previously published NMR results. Overall, our results offer valuable guidance for selecting optimal spin labels for 19F ENDOR distance measurement in proteins.

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用于 19F ENDOR 测距的自旋标签:分辨率、灵敏度和距离可预测性
19F 电子-核双共振(ENDOR)已成为确定生物大分子在 0.7-2 nm 范围内距离分布的一种极具吸引力的方法,而脉冲电子偶极光谱法却难以达到这一范围。19F ENDOR 方法依赖于自旋标记,在这项工作中,我们比较了各种标记的性能。我们用亚硝基、三丁基自由基和 Gd(III)螯合物在同一位点标记了 GB1 和泛素的四个含氟残基的蛋白质变体。此外,GB1 的双组氨酸变体也用 Cu(II) nitrilotriacetic acid 螯合物进行了标记。ENDOR测量是在W波段(95 GHz)进行的,在该波段中,19F信号与1H信号有很好的分离。观察到灵敏度存在差异,钆(III)螯合物的信噪比最高。不含甲基的新型三苯甲基标签 OXMA 显示出足够长的相位记忆时间,从而产生可接受的灵敏度。不过,这种标签的系链较长,有效地减少了 19F 与自旋标记位点 C 之间的最大可及距离。亚硝基和铜(II)标签通过取向选择提供了宝贵的额外几何见解。根据蛋白质的已知结构预测的电子核距离与 Gd(III)标签的实验值最为接近,而 Cu(II)标签 GB1 的电子核距离与之前公布的 NMR 结果非常一致。总之,我们的研究结果为蛋白质中 19F ENDOR 距离测量选择最佳自旋标签提供了有价值的指导。
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来源期刊
Physical Chemistry Chemical Physics
Physical Chemistry Chemical Physics 化学-物理:原子、分子和化学物理
CiteScore
5.50
自引率
9.10%
发文量
2675
审稿时长
2.0 months
期刊介绍: Physical Chemistry Chemical Physics (PCCP) is an international journal co-owned by 19 physical chemistry and physics societies from around the world. This journal publishes original, cutting-edge research in physical chemistry, chemical physics and biophysical chemistry. To be suitable for publication in PCCP, articles must include significant innovation and/or insight into physical chemistry; this is the most important criterion that reviewers and Editors will judge against when evaluating submissions. The journal has a broad scope and welcomes contributions spanning experiment, theory, computation and data science. Topical coverage includes spectroscopy, dynamics, kinetics, statistical mechanics, thermodynamics, electrochemistry, catalysis, surface science, quantum mechanics, quantum computing and machine learning. Interdisciplinary research areas such as polymers and soft matter, materials, nanoscience, energy, surfaces/interfaces, and biophysical chemistry are welcomed if they demonstrate significant innovation and/or insight into physical chemistry. Joined experimental/theoretical studies are particularly appreciated when complementary and based on up-to-date approaches.
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