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：
10255189
负责人：
KLAUS GAWRISCH
金额：
$ 185.11万
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/10255189
关键词：
2-arachidonylglycerol Affinity Alcoholism Amino Acid Sequence Amino Acids Anabolism Arachidonic Acids Area Attention Biological Biological Assay Biological Process Brain Buffers CNR1 gene CNR2 gene Cannabinoids Cattle Cell Membrane Structures Cell membrane Centrifugation Charge Chemicals Chimeric Proteins Cholesterol Cleaved cell Collaborations Coupled Coupling Crystallization Crystallography Data Deposition Detergents Development Dimerization Disease Docosahexaenoic Acids Drug Targeting Enzymes Equilibrium Escherichia coli Family Fluorescence Spectroscopy Future GTP gamma S GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Goals Guidelines Health Hydrocarbons Immobilization Integral Membrane Protein Investigation Ireland Isotope Labeling Label Laboratories Lateral Length Ligand Binding Ligands Light Lipid Bilayers Lipid Binding Lipids Lipoproteins Liquid substance Mammals Membrane Membrane Proteins Micelles Molecular Neurons Neurosciences Neutrons Nuclear Nutritional Omega-6 Fatty Acids Peptide Signal Sequences Phase Phospholipids Photoreceptors Physiological Polyunsaturated Fatty Acids Preparation Proteins Pseudomonas aeruginosa Radioactive Recombinants Resolution Retina Rhodopsin Role Sample Size Sampling Scheme Serine Signal Pathway Signal Transduction Site Solid Structure Study Section Sucrose Surface Surface Plasmon Resonance Synaptosomes Techniques Therapeutic Tissues anandamide biophysical properties cannabinoid receptor cholesteryl hemisuccinate college 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 monoolein photoactivation polyunsaturated fat protein activation proteoliposomes receptor receptor function receptor structure function reconstitution retinal rods segregation signal peptidase 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 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. 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. To reduce the size of samples with precious isotopically labeled GPCR, we collaborate on application of dynamic nuclear polarization (DNP) techniques to our samples that raise sensitivity by one to two orders of magnitude. 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. In collaboration with the Laboratory of Martin Caffrey, Trinity College, Dublin, Ireland, we studying the structure of lipoprotein signal peptidase II (LspA), a membrane protein which is a key enzyme in Pseudomonas aeruginosa. Its function is to release signal peptides from prolipoproteins. The goal of the project is to gather structural information related to LspA inhibition and lipoprotein processing. Both solution-state NMR experiments on LspA in detergent micelles and solid-state NMR experiments on LspA reconstituted into a monoolein cubic phase are conducted. Spectra of both the apo form of LspA as well as the globomycin-bound form were obtained. 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，从而在生理上相关的条件下实现了该GPCR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。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。为了减少具有珍贵的同位素标记GPCR的样品的大小，我们在动态核极化（DNP）技术的应用中合作，在我们的样品中提高了一到两个数量级的敏感性。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。我们与爱尔兰都柏林三一学院的马丁·卡夫里（Martin Caffrey）实验室合作研究了脂蛋白信号肽酶II（LSPA）的结构，这是一种膜蛋白，是pseudomonas aeruginosa中的关键酶。它的功能是从催生蛋白中释放信号肽。该项目的目的是收集与LSPA抑制和脂蛋白加工有关的结构信息。在清洁胶束中的LSPA上进行了两种溶液状态NMR实验，在LSPA上进行了固态NMR实验，在LSPA上重构为Monoolein Cubic相。获得了LSPA的APO形式的光谱以及球霉素结合的形式。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。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Methods for recombinant expression and functional characterization of human cannabinoid receptor CB2.

人大麻素受体 CB2 的重组表达和功能表征方法。

DOI：
10.5936/csbj.201303011
发表时间：
2013
期刊：
Computational and structural biotechnology journal
影响因子：
6
作者：
Yeliseev,AlexeiA
通讯作者：
Yeliseev,AlexeiA

Stabilization of functional recombinant cannabinoid receptor CB(2) in detergent micelles and lipid bilayers.

DOI：
10.1371/journal.pone.0046290
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Vukoti K;Kimura T;Macke L;Gawrisch K;Yeliseev A
通讯作者：
Yeliseev A

Use of tandem affinity chromatography for purification of cannabinoid receptor CB₂.

使用串联亲和层析纯化大麻素受体 CBα。

DOI：
10.1007/978-1-4939-1034-2_9
发表时间：
2014
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Locatelli-Hoops,SilviaC;Yeliseev,AlexeiA
通讯作者：
Yeliseev,AlexeiA

The role of membrane curvature elastic stress for function of rhodopsin-like G protein-coupled receptors.

膜曲率弹性应力对视紫红质样 G 蛋白偶联受体功能的作用。

DOI：
10.1016/j.biochi.2014.10.011
发表时间：
2014
期刊：
Biochimie
影响因子：
3.9
作者：
Soubias,Olivier;Teague,WalterE;Hines,KirkG;Gawrisch,Klaus
通讯作者：
Gawrisch,Klaus

Elastic properties of polyunsaturated phosphatidylethanolamines influence rhodopsin function.

多不饱和磷脂酰乙醇胺的弹性特性影响视紫红质功能。

DOI：
10.1039/c2fd20095c
发表时间：
2013
期刊：
Faraday discussions
影响因子：
3.4
作者：
TeagueJr,WalterE;Soubias,Olivier;Petrache,Horia;Fuller,Nola;Hines,KirkG;Rand,RPeter;Gawrisch,Klaus
通讯作者：
Gawrisch,Klaus