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：
9563824
负责人：
KLAUS GAWRISCH
金额：
$ 178.27万
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/9563824
关键词：
2-arachidonylglycerol Affinity Agonist Alcoholism Amino Acid Sequence Amino Acids Anabolism Arachidonic Acids Attention Biological Biological Assay Biological Process Brain Buffers CNR1 gene CNR2 gene Cannabinoids Cattle Cell Membrane Structures Centrifugation Charge Chemicals Chimeric Proteins Cholesterol Cleaved cell Collaborations Coupled Coupling Crystallization Crystallography Darkness Data Deposition Detergents Development Dimerization Disease Docosahexaenoic Acids Drug Targeting Elasticity Equilibrium Escherichia coli Family Fluorescence Spectroscopy Future G-substrate GTP gamma S GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Goals Guidelines Health Hydrocarbons Hydrophobicity Immobilization Integral Membrane Protein Investigation Isotope Labeling Label Laboratories Lateral Length Ligand Binding Ligands Light Link Lipid Bilayers Lipid Binding Lipids Liquid substance Mammals Membrane Membrane Proteins Micelles Modeling Molecular Molecular Models Neurons Neurosciences Neutrons Nutritional Omega-6 Fatty Acids Phase Phosphatidylethanolamine Phospholipids Photoreceptors Physiological Polyunsaturated Fatty Acids Preparation Property Proteins Radial Radioactive Receptor Activation Recombinants Resolution Retinal Rhodopsin Role Sampling Scheme Serine Signal Pathway Signal Transduction Site Solid Stress Structure Study Section Sucrose Surface Surface Plasmon Resonance Synaptosomes Therapeutic Thick Tissues Transducin anandamide biophysical properties cannabinoid receptor cholesteryl hemisuccinate crystallinity density design dimer dodecyl maltoside drug of abuse experimental study insight interest interstitial light scattering maltose-binding protein member metarhodopsin I metarhodopsin II milligram millisecond molecular modeling molecular scale monolayer photoactivation polyunsaturated fat protein activation proteoliposomes receptor receptor function receptor structure function reconstitution retinal rods segregation simulation solid state nuclear magnetic resonance tool visual process

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 by chemical labeling of amino acids as well by specific isotopic labeling of amino acids are under development to achieve the desired spectral resolution for structural analysis by NMR as well as EPR. The goal of these studies is to determine structural differences as a function of ligands that are bound to the receptor. Dimerization of GPCR has emerged as an essential mechanism regulating GPCR biosynthesis, maturation, ligand binding, coupling with G protein and downstream signaling in cell-signaling pathways. However, determining the oligomeric state of a GPCR in a membrane is challenging. It was explored if small angle neutron scattering (SANS) is a suitable tool to study the state of GPCR oligomerization at functional conditions of the receptor. Experiments were conducted with protonated bovine rhodopsin reconstituted into a perdeuterated lipid matrix. This yields maximal neutron scattering length density contrast between lipid and protein. Incoherent scattering of neutrons was minimized by conducting experiments in perdeuterated buffer. It was observed that the state of rhodopsin oligomerization in a lipid matrix at lipid-to-protein molar ratios near 500/1 depends on the state of photoactivation of the receptor. While dark-adapted rhodopsin was monomeric, bleached rhodopsin formed mostly dimers. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. We develop strategies for preparation of functional, recombinant CB2 and immobilization at solid interfaces. The successful deposition of CB2 was demonstrated by surface plasmon resonance. 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. We continue our studies on biophysical properties of the lipid matrix that are important for function of integral membrane proteins. In collaboration with laboratories that conduct molecular simulations, we explored the internal structure of the liquid ordered phase that forms in the presence of high cholesterol concentrations in membranes. The liquid ordered phase of a mixture of cholesterol and two lipids was shown to be itself inhomogeneous. Lateral segregation within the phase is observed, with regions of hexagonally packed saturated chains separated by interstitial regions enriched in cholesterol and unsaturated chains. The observed substructure explains existing experimental data and provides a focus for future efforts aimed at understanding the molecular scale structure of cell membranes. This picture of the phase provides an explanation for a number of experimental results, most of them obtained by NMR, which have until now lacked a consistent description in terms of a molecular model.

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。通过开发氨基酸的特定同位素标记，通过化学标记对氨基酸的化学标记方案进行了特定的同位素标记方案正在开发中，以实现NMR和EPR的结构分析所需的光谱分辨率。这些研究的目的是确定与受体结合的配体的函数的结构差异。 GPCR的二聚化已成为调节GPCR生物合成，成熟，配体结合，与G蛋白偶联和细胞信号途径中下游信号传导的基本机制。但是，确定膜中GPCR的寡聚状态是具有挑战性的。探讨了小角度中子散射（SAN）是在受体功能条件下研究GPCR寡聚状态的合适工具。用质子化的牛Rhodopsin重构为perdeperer的脂质基质进行实验。这产生了脂质和蛋白质之间的最大中子散射长度对比度。通过在perdepererated缓冲液中进行实验，可以最大程度地减少中子的不一致散射。据观察，在500/1接近脂质与蛋白质摩尔比的脂质基质中的视紫红质寡聚状态取决于受体的光激活状态。当黑色适应的视紫红质是单体的，但漂白的视紫红质主要形成二聚体。关于CB2的结构和功能研究可能会受益于将纯化的受体和功能受体固定在受控密度的合适表面上，最好是均匀的方向。我们制定了准备功能，重组CB2和在固体界面上固定化的策略。表面等离子体共振证明了CB2的成功沉积。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。我们继续对脂质基质的生物物理特性研究，这对于整体膜蛋白的功能很重要。通过与进行分子模拟的实验室合作，我们探索了在膜中存在高胆固醇浓度的情况下形成液体排序相的内部结构。胆固醇和两种脂质的混合物的液体有序相被证明本身是不均匀的。观察到该相内的侧分离，六角形填充饱和链的区域被富含胆固醇和不饱和链的间质区域分离。观察到的子结构解释了现有的实验数据，并为未来的努力提供了重点，以了解细胞膜的分子尺度结构。该阶段的图表为许多实验结果提供了解释，其中大多数由NMR获得，到目前为止，这些结果在分子模型方面缺乏一致的描述。