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、纯化和三维结晶方面。电子结晶学是膜蛋白结构测定的一种有价值的替代方法。我们建议通过二维膜晶体的电子结晶学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，现在被确认为氯-质子反向转运体，以及Mloti环核苷酸门控钾通道MloKl。CIC-ECL的结晶学结构是存在的，但现有的数据不能建立关于反向转运体的功能和pH依赖的抑制的结论性模型。对于MloK1，只有环核苷酸结合域的结构可用。我们得到了有序的CIC-ECL的2D晶体，并用电子结晶学方法确定了中性pH条件下的膜包埋的3D结构。利用电子衍射和分子置换，我们将在酸性pH下确定结构。这些数据应该可以确定与pH依赖的激活相关的构象变化。我们还初步得到了MloKl的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了四重对称性，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性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膜蛋白结晶设备，它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、样本温度和样本浓度。我们将建造这台机器，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，特别是透析过程中样品浓度的变化，对2D膜蛋白结晶起主要作用。

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.