Electron Microscopy of Membrane Proteins

膜蛋白的电子显微镜

• 批准号: 7302407
• 负责人: HENNING STAHLBERG
• 金额: $ 25.96万
• 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/7302407
• 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）：膜蛋白对于健康和疾病至关重要，并且代表了深入研究工作的主要焦点。膜蛋白的结构测定面临障碍，主要是在表达、纯化和 3D 结晶方面。电子晶体学是膜蛋白结构测定的一种有价值的替代方法。我们建议通过 2D 膜晶体的电子晶体学对两种调节离子通道膜蛋白进行结构研究：大肠杆菌氯离子通道 CIC-ecl（现已鉴定为氯质子反向转运蛋白）和 M. Loti 环核苷酸门控钾通道 MloKl。 CIC-ecl 的晶体学结构存在，但现有数据不允许建立关于反向转运蛋白的功能和 pH 依赖性抑制的结论性模型。对于MloK1，只有环核苷酸结合结构域的结构是可用的。我们已经获得了 CIC-ecl 的高度有序的 2D 晶体，我们希望通过电子晶体学来确定中性 pH 下的膜嵌入 3D 结构。然后，我们将利用电子衍射和分子置换来确定酸性 pH 下的结构。这些数据应该能够确定与 pH 依赖性激活相关的构象变化。我们还有初步的 MloKl 二维晶体。这些晶体和我们的单颗粒 3D 电子显微镜分析似乎表明四聚体通道结构与四重对称性存在偏差，这可能取决于环核苷酸的存在或不存在。我们将详细阐述该门控钾通道的膜嵌入3D结构，并研究其调节机制。我们还将开发一种新的 2D 膜蛋白结晶装置，该装置将允许控制迄今为止可同时访问的参数。这台新机器将在透析过程中测量溶液的浊度，并通过计算机反馈来控制透析速度、样品温度和样品浓度。我们将建造该机器并研究这些参数对二维晶体尺寸和质量的影响。我们的初步结果表明，这些参数，特别是透析过程中样品浓度的变化，在二维膜蛋白结晶中发挥着重要作用。