NMR Investigations Of Cell Membrane Structure

细胞膜结构的核磁共振研究

基本信息

批准号：
8559245
负责人：
KLAUS GAWRISCH
金额：
$ 206.51万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8559245
关键词：
2-arachidonylglycerol Affinity Agonist Alcoholism Amino Acid Sequence Arachidonic Acids Attention Biological Assay Biological Process Brain CNR1 gene CNR2 gene Cannabinoids Cattle Cell Membrane Structures Centrifugation Charge Chimeric Proteins Cholesterol Cleaved cell Coupled Crystallization Crystallography Data Detergents Disease Docosahexaenoic Acids Drug Delivery Systems Elasticity Equilibrium Escherichia coli Family Fatty Acids Fluorescence Spectroscopy GTP gamma S GTP-Binding Proteins Goals Guidelines Health Hydrocarbons Investigation Label Length Ligand Binding Ligands Light Link Lipid Bilayers Lipid Binding Lipids Liquid substance Mammals Membrane Membrane Proteins Micelles Modeling Molecular Neurons Neurosciences Nutritional Phase Phosphatidylethanolamine Phospholipids Photoreceptors Physiological Polyunsaturated Fatty Acids Preparation Property Proteins Radial Radioactive Receptor Activation Recombinants Resolution Retinal Rhodopsin Role Sampling Scheme Serine Simulate Site Solutions Stress Structure Study Section Sucrose Thick Tissues Transducin anandamide cannabinoid receptor cholesteryl hemisuccinate design dodecyl maltoside drug of abuse insight interest light scattering maltose-binding protein member metarhodopsin I metarhodopsin II milligram millisecond monolayer phosphatidylethanolamine photoactivation polyunsaturated fat protein activation proteoliposomes receptor receptor function receptor structure function reconstitution research study retinal rods solid state nuclear magnetic resonance visual process visual processing

项目摘要

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.

我们在大肠杆菌中重组表达CB2作为与麦芽糖结合蛋白和几个亲和力标签的融合。 CB2融合蛋白被溶解，纯化，裂解，并再次从切割产物中纯化CB2。我们广泛研究了洗涤剂，脂质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适合各种下游应用程序的功能受体制备和处理的准则。我们证明，需要有效稳定CB2（DDDM）CB2功能折叠的稳定性，需要一种阴离子胆固醇衍生物，胆固醇半盐酸酯（CHS）和高亲和大麻素的CP-55,940或SR-144,528的一致动作。与CHS相似，带有丝氨酸头组（PS）的带负电荷的磷脂在胶束中产生了显着的稳定作用，而未充电的磷脂无效。纯化的CB2重构为脂质双层保留了长达几周的功能，从而在生理相关条件下对该GPCR进行了高分辨率的结构研究。在毫克水平上重新建立了功能性CB2，并浓度为40微升，足以通过固态NMR进行结构研究。通过使用放射性配体的配体结合以及与2H-MAS NMR结合使用的氘化配体通过配体结合验证了受体的功能性，并通过在GTPGAMMAS放射性测定中使用重组产生的G蛋白进行了G蛋白激活研究。通过分析NMR，使用标记的脂质和CB2的荧光光谱，动态光散射以及蔗糖梯度离心研究了蛋白质脂质体的组成，大小和均匀性。在2 mg的13C和15N样品上进行的探索性NMR实验标记了CB2，并比较了实验结果与从CB2模型的原子坐标获得的模拟光谱的比较证明了实验概念的可行性。已经开发了特定的同位素标记方案，以实现结构分析的所需光谱分辨率。具有高含量的多不饱和磷脂酰乙醇胺（PE）的膜促进牛hodopopsin-II（MII）的形成，元霍顿II（MII）是激活G蛋白转丁蛋白的牛Rhopopsin的光介粒蛋白中间体。我们确定MII形成是否与PES的弹性特性进行了定量链接。多不饱和脂质的单层曲率弹性18：0-22：6N-3PE，18：0-22：5N-6PE和模型脂质18：1N-9-18：1N-9-18：1N-9-9PE在抗六边形相位。所有三种脂质均形成脂质单层，其曲率为26-28 Angstrom的自发半径相当低。含有高浓度多不饱和PE的膜中的负曲率弹性应激非常高。从受体周围的脂质层中释放了一小部分应力，足以将MI/MII平衡转移到激活G蛋白的状态MII。此外，多不饱和双层的疏水厚度约为27 a，已确定可匹配杜鹃丁物的疏水性跨膜螺旋的长度。数据表明，多不饱和脂质对于A类GPCR激活很重要，我们推测Rhodopsin模型与弱激动剂的GPCR和激活激活特别相关。