NMR Investigations Of Cell Membrane Structure

我们在大肠杆菌中以麦芽糖结合蛋白和几个亲和标签的融合形式重组表达了CB2。CB2-融合蛋白被溶解、纯化、融合裂解，并从裂解产物中再次纯化CB2。我们广泛研究了洗涤剂、脂类和大麻素配体对重组大麻素受体CB2稳定性的影响。这项工作导致了适用于广泛下游应用的全功能受体的制备和处理指南。我们证明了阴离子胆固醇衍生物胆固醇半琥珀酸酯(CHS)和高亲和力的大麻素配体CP-55,940或SR-144,528的协同作用是有效稳定CB2在十二烷基麦芽糖苷(DDM)/CHAPS洗涤剂溶液中功能折叠所必需的。与CHS类似，带有丝氨酸头基的带负电荷的磷脂在胶束中具有显著的稳定作用，而不带电荷的磷脂则不起作用。重组成脂质双层的纯化的CB2保留了长达几周的功能，从而能够在生理相关的条件下对这种GPCR进行高分辨率的结构研究。 已经实现了功能CB2在毫克水平上的重组，并浓缩到40微升的体积，足以用于固体核磁共振的结构研究。结合~2H-MAS核磁共振和重组产生的G蛋白在GTP-GammaS放射分析中的G蛋白活化研究，用放射性配体和氚配体结合来验证该受体的功能。用分析核磁共振、标记脂质和CB2荧光光谱、动态光散射和蔗糖梯度离心法研究了蛋白质脂质体的组成、大小和均一性。对均匀标记的13C和15N标记的CB2的2 mg样品进行了探索性的核磁共振实验，并将实验结果与根据CB2模型的原子坐标获得的模拟光谱进行了比较，证明了实验概念的可行性。为了达到结构分析所需的光谱分辨率，已经开发了特定的同位素标记方案。 含有高含量多不饱和磷脂酰乙醇胺(PE)的膜促进了变紫红质-II(MII)的形成，MII是牛视紫质的光中间体，激活G蛋白转导蛋白。我们确定了混合结构是否与PE的弹性性质有定量的联系。研究了多不饱和脂18：0-22：6N-3PE、18：0-22：5N-6PE和模型脂18：1N-9-18：1N-9PE单层膜在反六方相中的曲率弹性。这三种脂类都形成了自发曲率半径为26-28埃的脂单分子层。在含有高浓度多不饱和聚乙烯的膜中，负曲率弹性应力非常高。即使这种压力的一小部分从受体周围的脂层中释放出来，也足以将MI/MII平衡转向MII，即激活G蛋白的状态。此外，多不饱和双层膜的疏水厚度约为27A，这已被确定为与视紫红质的疏水跨膜螺旋的长度相匹配。这些数据表明，多不饱和脂类对A类gpr的激活是重要的，我们推测视紫红质模型与gpr的构成活性和弱激动剂的激活特别相关。

We express CB2 recombinantly in Escherichia coli as a fusion with maltose-binding protein and several affinity tags. The CB2-fusion protein is solubilized, purified, the fusion cleaved, and CB2 purified again from cleavage products. We extensively studied the effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2. The effort resulted in guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/ CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions. Reconstitution of functional CB2 at the level of milligrams, and concentration to a volume of 40 microliters, sufficient for structural studies by solid state NMR has been achieved. Functionality of the receptor was verified by ligand binding using radioactive ligands as well as deuterated ligands in combination with 2H-MAS NMR and by G protein activation studies using recombinantly produced G protein in a GTPgammaS radioactive assay. Composition, size, and homogeneity of proteoliposomes were investigated by analytical NMR, fluorescence spectroscopy using labeled lipid and CB2, dynamic light scattering, and sucrose gradient centrifugation. Exploratory NMR experiments conducted on a 2-mg sample of homogeneously 13C- and 15N labeled CB2 and comparison of experimental results with simulated spectra obtained from the atomic coordinates of a CB2 model have demonstrated feasibility of the experimental concept. Specific isotopic labeling schemes have been developed to achieve the desired spectral resolution for a structural analysis. Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (MII), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether MII-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18:0-22:6n-3PE, 18:0-22:5n-6PE and the model lipid 18:1n-9-18:1n-9PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 Angstrom. Negative curvature elastic stress in membranes containing high concentrations of polyunsaturated PEs is very high. Release of even a small fraction of this stress from the layer of lipids surrounding the receptor is sufficient to shift the MI/MII equilibrium towards MII, the state that activates G protein. Furthermore, polyunsaturated bilayers have a hydrophobic thickness of about 27 A which has been determined to match the length of the hydrophobic transmembrane helices of rhodopsin. The data show that polyunsaturated lipids are important for class A GPCR activation, and we speculate that the rhodopsin model is particularly relevant for constitutive activity of GPCR and activation by weak agonists.