MASS SPECTROMETRIC STUDIES OF INTEGRAL MEMBRANE PROTEINS & ION CHANNELS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们正在努力开发质谱学的用途，以帮助定义完整的膜蛋白和(特别是离子通道)在原子分辨率下使用衍射方法。D.A.Doyle、J.M.Cabral、R.A.Pfuetzner、A.Kuo、J.M.Gulbis、S.L.Cohen、B.T.Chait、R.MacKinnon在《科学》280，(1998)69-77《钾通道的结构：钾传导和选择性的分子基础》一书中发表了许多出版物。麦金农，S.L.科恩，A.Kuo，A.Lee，B.T.Chait，《原核生物和真核生物钾通道的结构保护》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结构域家族的定义》Cell 95(1998)649-655。J·M·古尔比斯、M·周、S·曼、R·麦金农：《细胞质的结构(电压依赖性钾通道的亚单位-T1组装)》，科学289(2000)123-127。M.Cadene，B.T.Chait，《用于整体膜蛋白的质谱分析的强健的洗涤剂友好型》，肛门。化学。72(2000)5655-5658。S.L.Cohen，B.T.Chait，《作为研究蛋白质结构的工具的质谱学》。生物物理学与生物分子结构综述30(2001)67-85。R.duzler，ER Campbell，M.Cadene，B.T.Chait，R.MacKinnon《ClC氯通道在3.0A下的X射线结构》揭示了阴离子选择性的分子基础：自然415(2002)287-94。江亚勇，李亚平，陈军，M.Cadene，B.T.Chait，R.MacKinnon：“钙门控钾通道的晶体结构和机制”，自然杂志417(2002)515-522。姜勇，李亚平，陈军，M.Cadene，B.T.Chait，R.麦金农：《钾通道的开孔构象》，自然417(2002)523-526。姜勇，A.Lee，J.Chen，V.Ruta，M.Cadene，B.T.Chait&Roderick MacKinnon：“电压相关K通道的X射线结构”，自然423(2003)33-41。最近的工作涉及Kir3.1K()通道，它通过G蛋白和脂质信号通路参与心率控制和神经元兴奋性。在大肠杆菌中的表达是通过将四分之三的跨膜孔替换为原核生物KIR通道的孔，留下来自Kir3.1的细胞质孔和膜界面区。在2.2A处确定了两个结构，选择性过滤器在测量误差(r.m.s.d.&lt；0.2A)内与变铅青链霉菌K()通道相同，表明K()选择性需要极端保守的三维结构。多个K()离子驻留在孔内，有助于解释电压依赖性的镁(2)和多胺的阻塞和强烈的整流。在内螺旋束和细胞质孔顶端的两个收缩可以起到门的作用：在一个结构中，顶端是开放的，在另一个结构中，顶端是闭合的。顶端的门控是通过细胞质孔亚单位的刚体运动来调节的。磷脂酰肌醇4，5-二磷酸相互作用残基提示了信号脂调节细胞质孔的可能机制。

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.