Sub-Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing Cu II -labelling of Double-Histidine Motifs with Lower Temperature
Sub-Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing Cu II -labelling of Double-Histidine Motifs with Lower Temperature
复制标题
亚微摩尔脉冲偶极 EPR 光谱揭示了双组氨酸基序的 Cu II 标记在较低温度下的增加
DOI：
10.1002/ange.201904848
复制
发表时间：
2019
影响因子：
--
通讯作者：
Wort J
中科院分区：
文献类型：
--
作者：
Wort J
电子顺磁共振（EPR）距离测量通过提供高度精确的几何约束，对生物分子的研究做出了越来越重要的贡献。将双组氨酸基序与CuIIspin标记组合可以进一步提高距离测量的精度。它也适用于含有可干扰巯基特异性标记的必需半胱氨酸的蛋白质。然而，非共价Cu II配位方法易受低结合亲和力的影响。本文中，解离常数（KD）直接从弛豫诱导偶极调制增强（RIDME）EPR实验的调制深度研究。这揭示了低温下EPR距离测量条件下的低至亚μ mCuIIKD。我们证明了利用双组氨酸基序用于EPR应用的可行性，即使在亚μ m蛋白质浓度下，在正交标记的CuII-氮氧化物系统中使用商业Q带EPR仪器。
Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with CuIIspin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent CuIIcoordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (KD) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μmCuIIKDs under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μmprotein concentrations in orthogonally labelled CuII–nitroxide systems using a commercial Q‐band EPR instrument.