Electron Microscopy of Membrane Proteins

膜蛋白的电子显微镜

• 批准号: 7476520
• 负责人: HENNING STAHLBERG
• 金额: $ 27.62万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476520
• 关键词: Binding; Biological Process; Cells; Chloride Channels; Chloride Ion; Chlorides; Collaborations; Computers; Crystallization; Crystallography; Cyclic Nucleotides; Data; Dependence; Devices; Dialysis procedure; Disease; Drug Delivery Systems; Electron Microscopy; Electrons; Escherichia coli; Face; Feedback; Health; Image; Ion Channel; Maps; Measures; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Monitor; Nucleotides; Numbers; Phospholipids; Physical Dialysis; Play; Potassium Channel; Production; Proteins; Protocols documentation; Protons; Regulation; Research; Research Personnel; Role; Sampling; Solutions; Speed; Structure; Temperature; Time; Transmission Electron Microscopy; United States National Institutes of Health; X-Ray Crystallography; antiporter; electron crystallography; insight; method development; particle; reconstitution; size; three dimensional structure; two-dimensional

DESCRIPTION (provided by applicant): Membrane proteins are central to health and disease, and represent the major focus of intensive research efforts. Structure determination of membrane proteins faces hurdles, mostly in expression, purification, and 3D crystallization. Electron crystallography represents a valuable alternative for the structure determination of membrane proteins. We propose to structurally study two regulated ion channel membrane proteins by electron crystallography of 2D membrane crystals: The E. coli chloride channel CIC-ecl, now identified as chloride-proton antiporter, and the M. loti cyclic nucleotide gated potassium channel MloKl. A crystallography structure of CIC-ecl exists, but the available data do not allow establishing a conclusive model about the functioning and pH-dependent inhibition of the antiporter. For MloKl only a structure of the cyclic nucleotide binding domain is available. We have obtained excellently ordered 2D crystals of CIC-ecl, with which we want to determine the membrane-embedded 3D structure at neutral pH by electron crystallography. Using electron diffraction and molecular replacement, we will then determine the structure at acidic pH. These data should allow determining the conformational changes associated with pH-dependent activation. We also have preliminary 2D crystals of MloKl. These crystals and our single particle 3D electron microscopy analysis appear to suggest a deviation of the tetrameric channel structure from four-fold symmetry, which might be dependent on the presence or absence of cyclic nucleotides. We will elaborate the membrane- embedded 3D structure of this gated potassium channel, and study the mechanism of its regulation. We will also develop a new 2D membrane protein crystallization device, which will allow controlling parameters that so far are simultaneously accessible. This new machine will measure the solution turbidity during dialysis, and via computer feedback will control the dialysis speed, sample temperature and sample concentration. We will construct that machine and study the influence of these parameters on 2D crystal size and quality. Our preliminary results indicate that these parameters, especially changes in the sample concentration during dialysis, play a major role in 2D membrane protein crystallization.

描述（由申请人提供）：膜蛋白是健康和疾病的核心，是密集研究工作的主要重点。膜蛋白面对障碍的结构测定主要是表达，纯化和3D结晶。电子晶体学代表了膜蛋白结构测定的宝贵替代方法。我们建议通过2D膜晶体的电子晶体学研究两种调节的离子通道膜蛋白：大肠杆菌氯化物通道CIC-ECL，现在被鉴定为氯化物 - 普罗替型抗植物和M. loti Cyclic cyclic cilleotide nucleotide god核苷酸盖型核苷酸门托库库克斯通道Mlokl。存在CIC-ECL的晶体学结构，但是可用的数据不允许建立有关抗毒剂的功能和pH依赖性抑制的结论模型。对于mlokl，仅可用循环核苷酸结合结构域的结构。我们已经获得了CIC-ECL的2D晶体，我们希望通过电子晶体学在中性pH下确定膜上包裹的3D结构。使用电子衍射和分子置换，我们将确定酸性pH下的结构。这些数据应允许确定与pH依赖性激活相关的构象变化。我们也有mlokl的初步2D晶体。这些晶体和我们的单个粒子3D电子显微镜分析似乎表明四重对称性的四聚体通道结构偏离，这可能取决于循环核苷酸的存在或不存在。我们将详细说明该门控钾通道的膜嵌入的3D结构，并研究其调节的机制。我们还将开发一个新的2D膜蛋白质结晶装置，该设备将允许控制到目前为止可以同时访问的参数。这台新机器将在透析过程中测量溶液浊度，并且通过计算机反馈将控制透析速度，样品温度和样品浓度。我们将构建该机器并研究这些参数对2D晶体尺寸和质量的影响。我们的初步结果表明，这些参数，尤其是透析过程中样品浓度的变化，在2D膜蛋白结晶中起主要作用。