Study of membrane protein structure by NMR spectroscopy: the KcsA channel

已经开发了一组基于TROSY-HNCO的3D实验，用于测量大的膜相关蛋白质中的15 N弛豫参数，其特征在于缓慢的翻滚时间和显著的光谱重叠。测量骨干15 N R1，R1 rho，15 N-1H NOE，和15 N CSA/偶极交叉相关已被证明和应用于研究的SDS胶束中的同源四聚体KcsA钾通道的动力学行为的条件下，该通道是在关闭状态。胶束包裹的跨膜结构域，KcsATM，被发现表现出高度的秩序，翻滚作为一个扁圆椭球体的全球旋转相关时间，tc = 38 - 2.5纳秒，在50 ℃和扩散各向异性，D/D = 0.79 - 0.05，对应于纵横比a/B 1.4。KcsA的N-和C-末端细胞内片段表现出相当大的内部动力学（S2值在0.2-0.45范围内），但明显比非结构化的随机线圈所观察到的更有序。在这些领域的弛豫行为证实了C-末端螺旋的位置，并表明在SDS胶束中，这种两亲性螺旋不关联成一个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时间尺度上没有升高的骨架动力学，其选择K+离子进入通道。 我们还进行了NMR研究的单体亚基的通道（KcsAM），溶解在SDS胶束。化学位移，溶剂交换，骨干15 N弛豫和残余偶极耦合（RDC）的数据显示TM 1螺旋保持完整，但TM 2螺旋包含一个独特的扭结，这是受浓度无关，但pH值依赖性的构象交换微秒的时间尺度。以G99为中心的扭结区域先前涉及四聚体KcsA通道的门控。一个基于RDC的模型KcsAM在酸性pH值取向TM 1和两个螺旋段的扭结TM 2的配置让人想起的开放构象的通道。因此，状态之间的转换似乎是单体的固有能力，四聚体组装体对转换产生调节作用，从而使通道具有生理门控特征。

A set of TROSY-HNCO-based 3D experiments has been developed for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone 15N R1, R1rho, 15N-1H NOE, and 15N CSA/dipolar cross correlation has been 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, KcsATM, is found to exhibit a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tc = 38 2.5 ns, at 50 C and a diffusion anisotropy, D// / D┴ = 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 (S2 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. We also carried out an NMR study of the monomeric subunit of the channel (KcsAM), solubilized in SDS micelles. Chemical shift, solvent exchange, backbone 15N relaxation and residual dipolar coupling (RDC) data show the TM1 helix to remain intact, but the TM2 helix contains a distinct kink, which is subject to concentration-independent but pH-dependent conformational exchange on a microsecond time scale. The kink region, centered at G99, was previously implicated in gating of the tetrameric KcsA channel. An RDC-based model of KcsAM at acidic pH orients TM1 and the two helical segments of the kinked TM2 in a configuration reminiscent of the open conformation of the channel. Thus, the transition between states appears to be an inherent capability of the monomer, with the tetrameric assembly exerting a modulatory effect upon the transition which gives the channel its physiological gating profile.