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

通过 NMR 波谱研究膜蛋白结构：KcsA 通道

• 批准号: 7593482
• 负责人: Ad - Bax
• 金额: $ 32.52万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593482
• 关键词: Behavior; Biological Models; C-terminal; Chemicals; Condition; Coupling; Data; Drug Delivery Systems; Encapsulated; Exhibits; Helix (Snails); Ions; Ligand Binding; Measurement; Measures; Membrane Proteins; Micelles; Modeling; Molecular Conformation; NMR Spectroscopy; Physiological; Positioning Attribute; Potassium Channel; Range; Relaxation; Residual state; Screening procedure; Solutions; Solvents; Structure; Technology; Time; Transmembrane Domain; Vertebral column; base; diffusion anisotropy; ear helix; milligram; monomer; nuclear Overhauser enhancement; protein structure; research study

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.

已经开发了一组基于TROSY-HNCO的3D实验，用于测量与膜相关的大型，膜相关蛋白的15N弛豫参数，其特征在于缓慢的翻滚时间和明显的光谱重叠。已经证明了主链15N R1，R1RHO，15N-1H NOE和15N CSA/偶极跨相关性，并应用于研究该通道处于封闭状态的条件下SDS胶束中同型KCSA钾通道的动态行为。发现胶束包裹的跨膜结构域KCSATM表现出高度的顺序，以全球旋转相关时间为注的椭圆形，TC = 38 2.5 ns，在50 c和扩散性各向异性，D /// d/ d = 0.79 0.05 = 0.79 0.05 = 0.79 0.05，对应于上方a// b的1.4 a/ b c。 KCSA的N-和C末端细胞内片段表现出相当大的内部动力学（在0.2-0.45范围内的S2值），但比对于非结构化的随机线圈所观察到的明显有序。这些结构域中的松弛行为证实了C末端螺旋的位置，并表明在SDS胶束中，该两亲性螺旋不会将其连接到稳定的同型螺旋螺旋束中。放松数据表明，对于5个残留选择性滤波器，在PS-NS时间尺度上没有升高的主链动力学，该滤波器选择K+离子进入通道。 我们还对通道（KCSAM）的单体亚基进行了NMR研究，并在SDS胶束中溶解。化学位移，溶剂交换，骨干15N松弛和残留偶极耦合（RDC）数据显示，TM1螺旋保持完整，但是TM2螺旋构成了一个独特的扭结，该扭结符合浓度独立但pH依赖性的pH依赖性构型在微秒时尺度上。以G99为中心的扭结区以前与四聚体KCSA通道的门控有关。基于酸性pH的KCSAM的基于RDC的模型TM1和扭结TM2的两个螺旋段，以使人联想到通道的开放构象。因此，状态之间的过渡似乎是单体的固有能力，四聚体组件对过渡产生了调节作用，这使该通道赋予了其生理门控谱。