Pulsed electron-electron double-resonance determination of spin-label distances and orientations on the tetrameric potassium ion channel KcsA.
Pulsed electron-electron double-resonance determination of spin-label distances and orientations on the tetrameric potassium ion channel KcsA.
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DOI：
10.1021/ja904808n
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发表时间：
2009-10-28
影响因子：
15
通讯作者：
Prisner, Thomas F.
中科院分区：
文献类型：
--
作者：
Endeward, Burkhard;Butterwick, Joel A.;MacKinnon, Roderick;Prisner, Thomas F.
脉冲电子-电子双共振（PELDOR）测量提出了钾离子通道KcsA溶解在洗涤剂和脂质中重组。利用MTSL对该蛋白的R64C突变体进行定点自旋标记。在两种磁场强度（0.3 T / x波段和1.2 T / q波段）和不同探针频率下，通过PELDOR实验确定了四聚体中自旋标签的取向。PELDOR数据的定量模拟支持一种强烈受限的氮氧化物，相对于中心通道轴呈65度角。一般来说，与可溶性蛋白或洗涤剂溶解样品相比，从膜重构制剂中获得的PELDOR数据质量较差。其中一个原因是氮氧化物的横向自旋弛豫时间T2减少，这是由于膜内四聚体的拥挤，即使在低蛋白脂比下也会发生。这种减少的T2可以通过重组未标记和标记蛋白的混合物来克服，从而产生高质量的PELDOR数据。在洗涤剂和膜重构制剂中观察到相同的PELDOR振荡频率及其对探针频率的依赖关系，这表明在两种环境中自旋标签的位置和取向是相同的。
Pulsed Electron-Electron Double Resonance (PELDOR) measurements are presented from the potassium ion channel KcsA both solubilized in detergent and reconstituted in lipids. Site-directed spin labeling using MTSL was performed with a R64C mutant of the protein. The orientations of the spin labels in the tetramer were determined by PELDOR experiments performed at two magnetic field strengths (0.3 T / X-band and 1.2 T / Q-band) and variable probe frequency. Quantitative simulation of the PELDOR data supports a strongly restricted nitroxide, oriented at an angle of 65 degrees relative to the central channel axis. In general, poorer quality PELDOR data was obtained from membrane-reconstituted preparations compared to soluble proteins or detergent-solubilized samples. One reason for this is the reduced transverse spin relaxation time T2 of nitroxides due to crowding of tetramers within the membrane that occurs even at low protein to lipid ratios. This reduced T2 can be overcome by reconstituting mixtures of unlabeled and labeled proteins, yielding high-quality PELDOR data. Identical PELDOR oscillation frequencies and their dependencies on the probe frequency were observed in the detergent and membrane-reconstituted preparations indicating that the position and orientation of the spin labels are the same in both environments.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
8
作者：
Guo, Zhefeng;Cascio, Duilio;Hubbell, Wayne L.
通讯作者：
Hubbell, Wayne L.
影响因子：
2.2
作者：
Chiang, YW;Borbat, PP;Freed, JH
通讯作者：
Freed, JH
影响因子：
16.6
作者：
Godt, Adelheid;Schulte, Miriam;Jeschke, Gunnar
通讯作者：
Jeschke, Gunnar
影响因子：
1.7
作者：
Margraf, D.;Bode, B. E.;Prisner, T. F.
通讯作者：
Prisner, T. F.