dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu2+ Labels
dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu2+ Labels
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DOI：
10.1021/acs.jpclett.1c01002
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发表时间：
2021-05-12
影响因子：
5.7
通讯作者：
Saxena, Sunil
中科院分区：
文献类型：
--
作者：
Casto, Joshua;Mandato, Alysia;Saxena, Sunil
最近，定点Cu 2+标记已成为一种深刻的生物物理工具，可以直接报告与蛋白质和核酸的结构、构象转变和动力学相关的距离限制。然而，Cu（2+）标记固有的短相记忆时间将可测量的距离限制在4-5 nm。在这项工作中，我们系统地研究了不同的方法来抑制电子-核和电子-电子耦合相互作用，以减少快速弛豫。我们表明，使用Cu 2+自旋浓度高达约。800 μ M对弛豫具有不变的影响，并且增加冷冻保护剂浓度降低了溶剂质子对弛豫的贡献。另一方面，蛋白质和溶剂的氘化使偶极调制信号的持续时间显著增加6倍以上至32 μ s。基于信号寿命的增加，可以使用Cu 2+标记测量高达9 nm及以上的距离。
Recently, site-directed Cu2+ labeling has emerged as an incisive biophysical tool to directly report on distance constraints that pertain to the structure, conformational transitions, and dynamics of proteins and nucleic acids. However, short phase memory times inherent to the Cu(2+ )labels limit measurable distances to 4-5 nm. In this work we systematically examine different methods to dampen electron-nuclear and electron- electron coupled interactions to decrease rapid relaxation. We show that using Cu2+ spin concentrations up to ca. 800 mu M has an invariant effect on relaxation and that increasing the cryoprotectant concentration reduces contributions of solvent protons to relaxation. On the other hand, the deuteration of protein and solvent dramatically increases the duration of the dipolar modulated signal by over 6-fold to 32 mu s. Based on this increase in signal longevity, distances up to 9 nm and beyond can potentially be measured with Cu2+ labels.