Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q-Band DEER EPR Spectroscopy.
Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q-Band DEER EPR Spectroscopy.
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DOI：
10.1002/anie.201609617
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发表时间：
2016-12-19
影响因子：
16.6
通讯作者：
Clore, G. Marius
中科院分区：
文献类型：
--
作者：
Schmidt, Thomas;Walti, Marielle A.;Baber, James L.;Hustedt, Eric J.;Clore, G. Marius
目前使用双电子-电子共振（DEER） EPR光谱测量多聚体质子化蛋白上自旋标记之间的距离通常限制在15-60 Å范围内。在这里，我们展示了如何将DEER实验扩展到~80µs的偶极进化时间，允许在多聚体蛋白中访问高达170 Å的距离。该方法依靠稀疏自旋标记，辅以蛋白质和溶剂的氘化，以最大限度地减少多自旋效应的有害影响，并大大增加表观自旋标记相记忆弛豫时间，并辅以q波段测量提供的高灵敏度。我们以四联体分子机器GroEL为例说明了这种方法。两个表面暴露的工程突变体R268C和E315C分别用于测量GroEL的两个七聚体环内和环间的两两距离分布，其平均值分别为20至100 Å和30至160 Å。测量的距离分布与已知的apo GroEL晶体结构一致。这里提出的方法应该显著扩大使用鹿的结构表征的构象变化在高阶低聚物。对称的稀疏氮氧化物自旋标记，完全氘化的多聚体蛋白质，延长了氮氧化物自旋标签之间的DEER距离测量高达170 Å。这是通过增加相记忆弛豫慢分量的长度和幅度来实现的，从而允许DEER数据被收集到很长的偶极演化时间（~80µs）。该方法在GroEL分子机器上得到了验证，GroEL分子机器由14个相同的亚基排列在两个七聚体环上。
Current distance measurements between spin-labels on multimeric protonated proteins using double electron-electron resonance (DEER) EPR spectroscopy are generally limited to the 15–60 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ~80 µs, permitting distances up to 170 Å to be accessed in multimeric proteins. The method relies on sparse spin-labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin-label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q-band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface-exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Å and from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers. Sparse nitroxide spin-labeling of symmetric, fully deuterated multimeric proteins, extends DEER distance measurements between nitroxide spin labels up to 170 Å. This is achieved by increasing the length and amplitude of the slow component of phase memory relaxation, thereby permitting DEER data to be collected out to long dipolar evolution times (~80 µs). The approach is demonstrated on the molecular machine GroEL comprising 14 identical subunits arranged in two heptameric rings.
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影响因子：
15
作者：
Endeward, Burkhard;Butterwick, Joel A.;MacKinnon, Roderick;Prisner, Thomas F.
通讯作者：
Prisner, Thomas F.
旋转对称的同性恋者中的Peldor。
影响因子：
1.7
作者：
Giannoulis A;Ward R;Branigan E;Naismith JH;Bode BE
通讯作者：
Bode BE
影响因子：
2.9
作者：
Krivov, Georgii G.;Shapovalov, Maxim V.;Dunbrack, Roland L., Jr.
通讯作者：
Dunbrack, Roland L., Jr.
DOI：
10.1002/chem.201505143
发表时间：
2016-03-24
期刊：
Chemistry (Weinheim an der Bergstrasse, Germany)
影响因子：
--
作者：
Valera S;Ackermann K;Pliotas C;Huang H;Naismith JH;Bode BE
通讯作者：
Bode BE
影响因子：
3.3
作者：
Jeschke, Gunnar;Sajid, Muhammad;Godt, Adelheid
通讯作者：
Godt, Adelheid