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

开发了一套基于TROSY-HNCO的3D实验，用于测量大的膜相关蛋白质的15N驰豫参数，其特征是翻滚时间慢，光谱重叠显著。主链15N R1、R1Rho、15N-1HNOE和15N CsA/偶极互相关的测量已被证明，并用于研究KCSA钾通道在十二烷基硫酸钠胶束中处于闭合状态时的动态行为。胶束包裹的跨膜结构域KcsATM表现出高度的有序性，在50℃时以扁平椭球的形式翻滚，全球旋转相关时间TC=38 2.5 ns，扩散各向异性D/D=0.79 0.05，对应于长宽比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力学(S2值在0.2-0.45范围内)，但明显比观察到的非结构化随机线圈的有序性更强。在这些区域的松弛行为证实了C-末端螺旋的位置，并表明在十二烷基硫酸钠胶束中，这个两亲性螺旋不会缔合成稳定的同质四聚体螺旋束。弛豫数据表明，对于选择K+离子进入通道的5-残基选择性过滤器，在PS-ns时间尺度上没有升高的主干动力学。 我们还对溶解在十二烷基硫酸钠胶束中的通道单体亚单位(KcsAM)进行了核磁共振研究。化学位移、溶剂交换、主干15N驰豫和剩余偶极耦合(RDC)数据表明TM1螺旋保持完整，但TM2螺旋包含一个明显的扭结，这在微秒级上受到浓度无关但pH依赖的构象交换的影响。以G99为中心的扭结区先前被认为与四聚体KCSA通道的门控有关。在酸性pH下，基于RDC的KcsAM模型将TM1和扭结的TM2的两个螺旋片段定位在一种使人想起通道开放构象的配置中。因此，状态之间的转变似乎是单体的固有能力，四聚体组装对转变施加调制效应，从而使通道具有生理门控轮廓。

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.