Electron Microscopy of Membrane Proteins

点击翻译按钮获取中文摘要

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.