Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel
Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel
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DOI：
10.1007/s10858-006-9071-4
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发表时间：
2006-10-01
影响因子：
2.7
通讯作者：
Bax, Ad
中科院分区：
文献类型：
--
作者：
Chill, Jordan H.;Louis, John M.;Bax, Ad
提出了一组基于 TROSY-HNCO ( tHNCO) 的 3D 实验，用于测量大型膜相关蛋白中的 N-15 弛豫参数，其特征是缓慢的翻滚时间和显着的光谱重叠。演示了主链 N-15 R-1、R-1p、N-15-{H-1} NOE 和 N-15 CSA/偶极互相关的测量，并将其应用于研究 SDS 胶束中同四聚 KcsA 钾通道在该通道处于关闭状态的条件下的动态行为。胶束封装的跨膜结构域 KcsA (TM) 表现出高度有序性，作为扁椭球体翻滚，在 50°C 时具有全局旋转相关时间 tau(c) = 38 +/- 2.5 ns，并且具有扩散各向异性，D-平行于/D-垂直于 = 0: 79 +/- 0: 05，对应于纵横比a/b >= 1.4。 KcsA 的 N 端和 C 端细胞内片段表现出相当大的内部动态（S-2 值在 0.2 - 0.45 范围内），但明显比观察到的非结构化随机卷曲更加有序。这些结构域中的松弛行为证实了 C 端螺旋的位置，并表明在 SDS 胶束中，这种两亲性螺旋不会缔合成稳定的同源四聚体螺旋束。弛豫数据表明 5 残基选择性过滤器在 ps-ns 时间尺度上不存在升高的主链动力学，该过滤器选择 K+ 离子进入通道。
A set of TROSY-HNCO ( tHNCO)-based 3D experiments is presented for measuring N-15 relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone N-15 R-1, R-1p, N-15-{H-1} NOE, and N-15 CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsA (TM), exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tau(c) = 38 +/- 2.5 ns, at 50 degrees C and a diffusion anisotropy, D-parallel to/ D-perpendicular to = 0: 79 +/- 0: 05, corresponding to an aspect ratio a/b >= 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics ( S-2 values in the 0.2 - 0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C- terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps-ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel.