MASS SPECTROMETRIC STUDIES OF INTEGRAL MEMBRANE PROTEINS & ION CHANNELS

完整膜蛋白的质谱研究

• 批准号: 8169097
• 负责人: RODERICK MACKINNON
• 金额: $ 0.47万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169097
• 关键词: Anions; Anus; Biophysics; Calcium; Cells; Chloride Channels; Computer Retrieval of Information on Scientific Projects Database; Cytoplasmic Structures; Detergents; Escherichia coli; Family; Funding; GTP-Binding Proteins; Grant; Heart Rate; Institution; Integral Membrane Protein; Ion Channel; Ions; Left; Lipids; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Methods; Molecular; Molecular Conformation; Movement; Nature; Phosphatidylinositols; Polyamines; Potassium Channel; Publications; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Ruta; Science; Signal Pathway; Signal Transduction; Source; Streptomyces lividans; Structure; United States National Institutes of Health; Work; base; constriction; interfacial; neuronal excitability; protein structure; three dimensional structure; tool; voltage

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are making a major effort to develop the utility of mass spectrometry to assist in the definition of integral membrane proteins and (especially ion channels) at atomic resolutions using diffraction methods. A number of publications have resulted from this work D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, R. MacKinnon, "The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity" Science 280, (1998) 69-77. R. MacKinnon, S.L. Cohen, A. Kuo, A. Lee, B.T. Chait "Structural Conservation in Prokaryotic and Eukaryotic Potassium Channels" Science 280 (1998) 106-109. J.H. Morais Cabral, A. Lee, S. Cohen, B.T. Chait, M. Li, R. MacKinnon "Structure of the HERG potassium channel amino-terminal domain: definition of a structural family of PAS domains" Cell 95 (1998) 649-655. J.M. Gulbis, M. Zhou, S. Mann, R. MacKinnon "Structure of the Cytoplasmic ( Subunit-T1 Assembly of Voltage-Dependent K+ Channels" Science 289(2000) 123-127. M. Cadene, B.T. Chait "A Robust Detergent-Friendly for Mass Spectrometric Analysis of Integral Membrane Proteins" Anal. Chem. 72 (2000) 5655-5658. S.L. Cohen, B.T. Chait "Mass Spectrometry as a tool for Studying Protein Structure" Ann. Reviews of Biophysics and Biomolecular Structure 30 (2001) 67-85. R. Dutzler, ER Campbell, M. Cadene, B.T. Chait, R.MacKinnon "X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity" Nature 415 (2002) 287-94. Y. Jiang, A. Lee, J. Chen, M. Cadene, B. T. Chait, R. MacKinnon "Crystal structure and mechanism of a calcium-gated potassium channel" Nature 417 (2002) 515-522. Y. Jiang, A. Lee, J. Chen, M. Cadene, B. T. Chait, R. MacKinnon "The open pore conformation of potassium channels" Nature 417 (2002) 523-526. Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait & Roderick MacKinnon "X-ray structure of a voltage-dependent K+ channel" Nature 423 (2003) 33-41. The most recent work involves the Kir3.1 K(+) channel, which participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2 A. The selectivity filter is identical to the Streptomyces lividans K(+) channel within error of measurement (r.m.s.d.<0.2 A), suggesting that K(+) selectivity requires extreme conservation of three-dimensional structure. Multiple K(+) ions reside within the pore and help to explain voltage-dependent Mg(2+) and polyamine blockade and strong rectification. Two constrictions, at the inner helix bundle and at the apex of the cytoplasmic pore, may function as gates: in one structure the apex is open and in the other, it is closed. Gating of the apex is mediated by rigid-body movements of the cytoplasmic pore subunits. Phosphatidylinositol 4,5-biphosphate-interacting residues suggest a possible mechanism by which the signaling lipid regulates the cytoplasmic pore.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们正在大力开发质谱分析的实用性，以协助使用衍射方法以原子分辨率定义完整的膜蛋白和（特别是离子通道）。这项工作已出版了许多出版物 D.A.多伊尔，J.M. 卡布拉尔，R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L.科恩，B.T. Chait, R. MacKinnon，“钾通道的结构：K+ 传导和选择性的分子基础”，《科学》280，(1998) 69-77。 R.麦金农，S.L.科恩、A. Kuo、A. Lee、B.T. Chait“原核和真核钾通道的结构保守”，科学 280 (1998) 106-109。 J.H. Morais Cabral、A. Lee、S. Cohen、B.T. Chait，M. Li，R. MacKinnon“HERG 钾通道氨基末端结构域的结构：PAS 结构域结构家族的定义”Cell 95 (1998) 649-655。 J.M. Gulbis、M. Zhou、S. Mann、R. MacKinnon“细胞质的结构（电压依赖性 K+ 通道的亚基 T1 组装”）《科学》289(2000) 123-127。M. Cadene、B.T. Chait“用于对整体膜蛋白进行质谱分析的稳健的去污剂友好型”Anal. Chem。 72 (2000) 5655-5658。 S.L.科恩，B.T. Chait“质谱作为研究蛋白质结构的工具”Ann。生物物理学和生物分子结构评论 30 (2001) 67-85。 R. Dutzler、ER Campbell、M. Cadene、B.T. Chait, R.MacKinnon “3.0 A 下 ClC 氯离子通道的 X 射线结构揭示了 阴离子选择性” Nature 415 (2002) 287-94. Y. Jiang, A. Lee, J. Chen, M. Cadene, B. T. Chait, R. MacKinnon “钙门控钾通道的晶体结构和机制” Nature 417 (2002) 515-522. Y. Jiang, A. Lee, J. Chen, M. Cadene, B. T. Chait, R.麦金农“开放 钾通道的孔构象” Nature 417 (2002) 523-526。 Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait & Roderick MacKinnon “电压依赖性 K+ 通道的 X 射线结构” Nature 423 (2003) 33-41。最近的工作涉及 Kir3.1 K(+) 通道，它参与 通过 G 蛋白和脂质信号通路控制心率和神经元兴奋性。通过用原核 Kir 通道的孔替换四分之三的跨膜孔，保留 Kir3.1 起源的细胞质孔和膜界面区域，实现了在大肠杆菌中的表达。在 2.2 A 下确定了两种结构。选择性滤光片与 浅青紫链霉菌 K(+) 通道在测量误差范围内 (r.m.s.d.<0.2 A)，表明 K(+) 选择性需要三维结构的极度保守。孔内存在多个 K(+) 离子，有助于解释电压依赖性 Mg(2+) 和多胺阻断和强整流。两个收缩，在内螺旋束和细胞质的顶端 孔，可以起到门的作用：在一种结构中，顶端是开放的，而在另一种结构中，顶端是关闭的。顶端的门控是由细胞质孔亚基的刚体运动介导的。磷脂酰肌醇 4,5-二磷酸相互作用残基表明信号脂质调节细胞质孔的可能机制。