Electron Microscopy of Membrane Proteins

描述（由申请人提供）：膜蛋白是健康和疾病的核心，代表了密集研究工作的主要焦点。膜蛋白的结构测定面临着障碍，主要是在表达，纯化和3D结晶。电子晶体学代表了一个有价值的替代膜蛋白的结构测定。我们建议通过二维膜晶体的电子晶体学来研究两种受调控的离子通道膜蛋白的结构：E。coli的氯离子通道CIC-ecl，现已被鉴定为氯离子质子逆向转运蛋白;环核苷酸门控钾通道MloKl。CIC-ecl的晶体学结构存在，但现有的数据不允许建立一个决定性的模型的功能和pH依赖性抑制的反向转运蛋白。对于MloKl，仅可获得环核苷酸结合结构域的结构。我们已经获得了良好的有序的二维晶体的CIC-ecl，我们想确定的膜嵌入的三维结构在中性pH值的电子晶体学。使用电子衍射和分子置换，我们将确定在酸性pH值的结构。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粒子3D电子显微镜分析似乎表明四聚体通道结构偏离四重对称性，这可能取决于环核苷酸的存在或不存在。我们将详细阐述这种门控钾通道的膜包埋三维结构，并研究其调控机制。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浊度，并通过计算机反馈控制透析速度、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，特别是在透析过程中的样品浓度的变化，发挥了重要作用，在二维膜蛋白结晶。

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.