High-Resolution Structure of a Protein Spin-Label in a Solvent-Exposed β-Sheet and Comparison with DEER Spectroscopy
High-Resolution Structure of a Protein Spin-Label in a Solvent-Exposed β-Sheet and Comparison with DEER Spectroscopy
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DOI：
10.1021/bi300328w
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发表时间：
2012-08-14
期刊：
影响因子：
2.9
通讯作者：
Saxena, Sunil
中科院分区：
文献类型：
--
作者：
Cunningham, Timothy F.;McGoff, Marshall S.;Saxena, Sunil
X射线晶体学已经成为一个有用的工具，在发展中的定点自旋标记，解决旋转异构体的氮氧自旋标记侧链在各种α-螺旋环境。在这项工作中，解决了蛋白G的B1免疫球蛋白结合域（GB 1）的双自旋标记的N8 C/K28 C突变体的晶体结构。双突变体在结晶条件下形成结构域交换的二聚体。两个旋转异构体状态的自旋标签解决在溶剂暴露的α-螺旋网站，在残基28，这些是在良好的协议与旋转异构体先前报道的螺旋结构。第二个位点位于内部β链上的残基8处，显示存在三种不同的暴露于溶剂的侧链旋转异构体。这些旋转异构体中的一种在R1位点的晶体结构内很少观察到，这表明α-螺旋位点所共有的H-α和S-δ氢键在该内部β链残基处不存在。β-链位点的变温连续波（CW）实验显示了与晶体学中观察到的旋转异构体状态相关的两种不同组分。有趣的是，室温下的CW数据可以在不使用序参量的情况下拟合，这与缺乏H-α和S-δ相互作用一致。此外，对单体形式的GB 1双突变体进行了双电子电子共振（DEER）光谱分析，得到了最可能的自旋间距离为25 +/-1埃。为了评估测量的DEER距离的准确性，将在结构域交换的GB 1二聚体的晶体结构中观察到的旋转异构体建模为野生型单体GB 1的高分辨率结构。在所得到的GB 1结构模型中生成的距离与最可能的DEER距离在近似2埃的范围内匹配。结果是有趣的，因为它们通过直接实验测量表明，在该晶体中发现的R1的旋转异构态提供了与DEER测量的最可能距离非常接近的匹配
X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of alpha-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed a-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior beta-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the H-alpha and S-delta hydrogen bond that is common to alpha-helical sites is absent at this interior beta-strand residue. Variable temperature continuous wave (CW) experiments of the beta-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the H-alpha and S-delta interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 +/- 1 angstrom. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within similar to 2 angstrom. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER