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：
8941369
负责人：
KLAUS GAWRISCH
金额：
$ 173.92万
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/8941369
关键词：
2-arachidonylglycerol Affinity Agonist Alcoholism Amino Acid Sequence Arachidonic Acids Arginine Attention Biological Assay Biological Process Brain CNR1 gene CNR2 gene Cannabinoids Cattle Cell Membrane Structures Centrifugation Charge Chimeric Proteins Cholesterol Cleaved cell Collaborations Coupled Crystallization Crystallography Data Deposition Detergents Development Disease Docosahexaenoic Acids Drug Targeting Elasticity Equilibrium Escherichia coli Exhibits Family Fatty Acids Fluorescence Spectroscopy Future GTP gamma S GTP-Binding Proteins Goals Guidelines Health Hydrocarbons Immobilization Integral Membrane Protein Investigation Label Laboratories Lateral Length Ligand Binding Ligands Light Link Lipid Bilayers Lipid Binding Lipids Liquid substance Mammals Membrane Membrane Lipids Membrane Proteins Micelles Modeling Molecular Molecular Models National Institute of Neurological Disorders and Stroke Neurons Neurosciences Nutritional Phase Phosphatidylethanolamine Phospholipids Photoreceptors Physiological Play Polyunsaturated Fatty Acids Positioning Attribute Preparation Property Proteins Radial Radioactive Receptor Activation Recombinants Resolution Retinal Rhodopsin Roentgen Rays Role Sampling Scheme Serine Simulate Site Solid Solutions Stress Structure Study Section Sucrose Surface Surface Plasmon Resonance Thick Tissues Transducin Voltage-Gated Potassium Channel Well in self anandamide biophysical properties cannabinoid receptor cholesteryl hemisuccinate density design dodecyl maltoside drug of abuse 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 research study retinal rods segregation simulation solid state nuclear magnetic resonance visual process visual processing voltage

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 are under development to achieve the desired spectral resolution for structural analysis. 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. In collaboration with MPBS, NINDS we contribute to investigation of the voltage-gated potassium channel KvAP by conducting solid-state NMR experiments. Our experiments with the S1-S4 voltage sensing domain of the channel suggest that lipids have weak and transient interactions with the protein that do not detectably alter the structure of the domain. Data obtained on S1-S4 domain embedded in lipid membranes are remarkably consistent with both X-ray and solution NMR structures of the domain obtained in detergent micelles. The S1S4 domain exhibits extensive interactions with lipids and the domain is heavily hydrated when embedded in a membrane. There is evidence for some preferential interactions of anionic lipids with the S1S4 domain. The arginine residues within the S1S4 domain, which play a major role in voltage sensing, are well hydrated and are positioned in close proximity to lipids, exhibiting close interactions with both lipid headgroups and acyl chains. 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。正在开发特定的同位素标记方案，以实现所需的光谱分辨率进行结构分析。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。通过与MPBS合作，我们通过进行固态NMR实验来研究电压门控钾通道KVAP。我们对通道的S1-S4电压传感结构域进行的实验表明，脂质与未检测到的蛋白质具有弱和瞬时相互作用，而蛋白质无法检测到域的结构。在嵌入脂质膜中嵌入的S1-S4结构域获得的数据与在洗涤剂胶束中获得的域的X射线和溶液NMR结构都非常一致。 S1S4结构域与脂质表现出广泛的相互作用，当嵌入膜中时该结构域被大量水合。有证据表明，阴离子脂质与S1S4结构域的某些优先相互作用。 S1S4结构域中的精氨酸残基在电压传感中起主要作用，它具有充分的水合，并且位于与脂质的近距离接近，与脂质头组和酰基链均表现出密切相互作用。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。