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障碍，主要是在表达、纯化和三维结晶。电子晶体学是测定膜蛋白结构的一种有价值的方法。我们提出利用二维膜晶体的电子晶体学技术对两种受调控的离子通道膜蛋白进行结构研究：大肠杆菌氯离子通道CIC-ecl（现鉴定为氯离子质子反转运体）和洛蒂杆菌环核苷酸门控钾通道MloKl。CIC-ecl的晶体结构是存在的，但现有的数据不允许建立一个关于反转运蛋白功能和ph依赖性抑制的结论性模型。对于MloKl，只有环核苷酸结合域的结构是可用的。我们已经获得了有序的CIC-ecl二维晶体，我们想用电子晶体学来确定在中性pH下膜包埋的三维结构。利用电子衍射和分子替换，我们将确定在酸性ph下的结构。这些数据应该允许确定与ph依赖性激活相关的构象变化。我们也有初步的二维MloKl晶体。这些晶体和我们的单粒子三维电子显微镜分析似乎表明四聚体通道结构偏离了四重对称，这可能取决于环核苷酸的存在或缺失。我们将详细阐述这种门控钾通道的膜嵌入三维结构，并研究其调控机制。我们还将开发一种新的二维膜蛋白结晶装置，它将允许控制迄今为止可同时访问的参数。该仪器将测量透析过程中的溶液浊度，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起主要作用。

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.