Small Gd(III) Tags for Gd(III)–Gd(III) Distance Measurements in Proteins by EPR Spectroscopy
Small Gd(III) Tags for Gd(III)–Gd(III) Distance Measurements in Proteins by EPR Spectroscopy
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小 Gd(III) 标签，用于通过 EPR 光谱法测量蛋白质中的 Gd(III)–Gd(III) 距离
DOI：
10.1021/acs.inorgchem.8b00133
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发表时间：
2018
影响因子：
4.6
通讯作者：
Graham, Bim
中科院分区：
文献类型：
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作者：
Prokopiou, Georgia;Lee, Michael D.;Collauto, Alberto;Abdelkader, Elwy H.;Bahrenberg, Thorsten;Feintuch, Akiva;Ramirez-Cohen, Marie;Clayton, Jessica;Swarbrick, James D.;Graham, Bim
C7-Gd和C8-Gd标记物是基于环戊二烯的镧系元素标记物，用于与蛋白质中的半胱氨酸残基结合。标签是对映异构体，其不同之处在于配位镧系元素离子的2-羟丙基侧臂的构型。在这里，我们报告了电子顺磁共振（EPR）性能的C7-Gd（S构型）和C8-Gd（R构型）标签负载Gd（III）的两个突变体的同源二聚体ERp 29蛋白。发现W-波段EPR谱在自由状态下的标签和与蛋白质缀合后的标签之间不同。此外，光谱是敏感的标记的位置，这可能源于环境依赖的电荷密度上的Gd（III）-配位氧。这与先前用不同镧系元素离子进行的NMR实验一致，该实验表明对H-键合的敏感性。W-band 1H-ENDOR（电子-电子双共振）实验检测到的取向选择在中央过渡的影响，由于一个相对较窄的分布，如模拟所示，在的取向参数。与此相反，来自DEER（双电子-电子共振）测量的距离分布是不敏感的theR或S配置的标签，并没有表现出任何取向选择的影响。的DEER测量忠实地反映了不同的宽度的距离分布在不同的蛋白质位点的协议与以前的DEER测量使用其他Gd（III）标签。由于它们的小尺寸、短的蛋白质系链和宽的中心EPR跃迁，C7-Gd和C8-Gd标签是通过EPR光谱测量相对短（<4 nm）距离的有吸引力的Gd（III）标签。
TheC7-GdandC8-Gdtags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of theC7-Gd(Sconfiguration) andC8-Gd(Rconfiguration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band1H-ENDOR (electron–electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron–electron resonance) measurements were insensitive to theRorSconfiguration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, theC7-GdandC8-Gdtags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.