Nanometer-Scale Distance Measurements in Proteins Using Gd3+ Spin Labeling
Nanometer-Scale Distance Measurements in Proteins Using Gd3+ Spin Labeling
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DOI：
10.1021/ja1015662
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发表时间：
2010-07-07
影响因子：
15
通讯作者：
Goldfarb, Danielle
中科院分区：
文献类型：
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作者：
Potapov, Alexey;Yagi, Hiromasa;Goldfarb, Danielle
测量蛋白质中特定位点之间的纳米级距离的方法对于分析蛋白质的结构和功能是必不可少的。本文介绍了用Gd ~（3+）自旋标记高场脉冲电子顺磁共振（EPA）测量蛋白质中纳米级距离的方法。为了评价这种测量的性能，我们对两种蛋白质进行了四脉冲双电子电子共振（DEER）测量，p75 ICD和tau（C）14，在策略性选择的位点用两个氮氧自由基或两个Gd 3+自旋标记物标记。与使用氮氧自由基的常规定点自旋标记类似，将作为吡啶二羧酸衍生物的Gd 3+标签共价连接到半胱氨酸巯基上。（类似于9 5 GHz，0.35 T）和W波段（95 GHz，3.5 T）光谱仪用于氮氧自由基标记的蛋白质，W波段用于Gd 3+标记的蛋白质。在蛋白质p75 ICD中，发现两种氮氧自由基的取向实际上不相关，因此，在W波段和在X波段一样容易获得距离分布。测得的Gd 3 +-Gd 3+距离分布在2.9 nm处有一个最大值，相比2 - 5 nm的氮氧化物在蛋白质tau（C）14，然而，氮氧化物的取向相关，和W-波段测量表现出较强的取向选择，防止直接提取的距离分布。X-波段的测量给出了一个氮氧化物-氮氧化物的距离分布，最大值在2 - 5 nm，和W-波段的测量给出了一个Gd 3 +-Gd 3+的距离分布，最大值在3 - 4 nm。获得的Gd 3 +-Gd 3+距离分布与结构模型的预期吻合良好，该模型考虑了标签及其与半胱氨酸残基的连接的灵活性。这些结果表明，Gd ~（3+）标记是一种可行的高场距离测量技术，其特点是在蛋白质量方面比使用氮氧自由基自旋标记的X-波段脉冲EPR测量灵敏度提高一个数量级。
Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function In this work we introduce Gd3+ spin labeling for nanometer-range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPA) To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and tau(C)14, labeled at strategically selected sites with either two nitroxides or two Gd3+ spin labels In analogy to conventional site-directed spin labeling using nitroxides, Gd3+ tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups Measurements were carried out on X-band (similar to 9 5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd3+-labeled proteins In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd3+-Gd3+ distance distribution had a maximum at 2.9 nm, as compared to 2 5 nm for the nitroxides In the protein tau(C)14, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2 5 nm, and the W-band measurements gave a Gd3+-Gd3+ distance distribution with a maximum at 3 4 nm. The Gd3+-Gd3+ distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd3+-labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection