NMR Investigations Of Cell Membrane Structure

细胞膜结构的核磁共振研究

• 批准号: 7317629
• 负责人: KLAUS GAWRISCH
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 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/7317629
• 关键词: NMR; Investigations; Cell; Membrane; Structure

The objectives of this project are to: (i) study structure, dynamics, and domain formation in membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (ii) study the interaction of the polyunsaturated lipid matrix with G-protein coupled membrane receptors (GPCR), and (iii) investigate structure and function of selected GPCR with relevance for alcoholism in reconstituted membrane systems. (i) It was explored if the surface of the GPCR rhodopsin should be viewed as homogeneous and the surrounding membrane as a continuum, or if specific interactions, in particular with polyunsaturated lipids may play a role in rhodopsin activation. The association of rhodopsin with poly- and monounsaturated lipids was studied by 1H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing bovine rhodopsin and lipids with polyunsaturated, docosahexaenoyl (DHA) and monounsaturated oleoyl chains. Rhodopsin was photoactivated in the spinning MAS rotor and magnetization transfer followed as a function of time after photoactivation. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for meta-III rhodopsin. Rates of magnetization transfer from protein to DHA are lipid headgroup dependent and increased in the sequence PC, PS, PE. In experiments conducted as a function of DHA content, it was shown that poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rhodopsin-associated lipids are in fast exchange with lipids in the bulk of the matrix on a timescale of milliseconds or shorter. Deciphering of the functional consequences of specific lipid-rhodopsin associations requires further investigations. We characterized the properties of liquid ordered phases related to formation of so-called rafts in biomembranes. Although biochemical evidence for existence of rafts is strong, detection of the structural equivalent of a raft in biomembranes has proven to be extremely difficult. In experiments on well-characterized model membranes it was shown that 1H MAS NMR spectra of lipids in liquid ordered domains (lo) have a distinctly different linewidth from liquid disordered (ld) and solid ordered (so) membrane regions. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the lo phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/trans isomerization in lo. In spectra recorded as a function of temperature, the onset of lo domain (raft) formation is seen as a sudden onset of line broadening. The 1H MAS NMR method of lo phase detection does not require labeling and may be applied to rafts in cell membranes. Experiments on biological membranes are underway. (ii) We developed reagents and methods for formation of tubular single lipid bilayers containing high concentrations of GPCR that cover the pore walls of porous anodic aluminum oxide (AAO) filters. One square centimeter of the filter material with a thickness of 60 micrometers yields up to 500 cm2 of oriented membranes, which is sufficient for multinuclear solid state NMR studies on the lipid matrix and on incorporated, isotopically labeled protein. By solid state NMR methods we determined that the membranes are separated from the support by a closed and stable aqueous cushion. The inner surface of the lipid tubules is freely accessible from an outside solution. The aluminum oxide-based support provides the advantage of high flow rates to exchange solutions, efficient particle retention, rigid, uniform surface, and transparency (when wet). The GPCRs rhodopsin, purified from natural sources, as well as the recombinant peripheral cannabinoid receptor, CB2, expressed in E. coli were incorporated into the tubular bilayers in functional form. Reconstitution of bovine rhodopsin into AAO filters did not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-Vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTP-gamma-S binding assay. Lipid-rhodopsin interaction was investigated by 2H NMR on sn-1-, or sn-2 chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed spectral density of motions with correlation times in the range from nano- to microseconds, detected as significant reduction of spin-spin relaxation times. The change of lipid chain order parameters due to interaction with rhodopsin was insignificant. (iii) Human peripheral-type cannabinoid receptor (CB2) was expressed in E. coli as a fusion with the maltose-binding protein and thioredoxin. Successful expression of the full-length fusion was confirmed by Western-blot analysis and mass spectroscopy. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. Agonist stimulation of urea-treated E. coli membranes expressing recombinant CB2 resulted in an activation of the G proteins in the in vitro coupled assay. For purification of the protein, Strep-tags and decahistidine-tags were introduced at various locations in the polypeptide chain. The fusion-CB2 was successfully cleaved at Tev-protease cleavage sites and the CB2 purified by two steps of affinity chromatography. The purified CB2 was successfully reconstituted into lipid bilayers and retained, at least partially, its ligand binding properties. The protocol allows for expression and purification of milligram quantities of the recombinant receptor for structural studies by NMR and scattering techniques.

The objectives of this project are to: (i) study structure, dynamics, and domain formation in membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (ii) study the interaction of the polyunsaturated lipid matrix with G-protein coupled membrane receptors (GPCR), and (iii) investigate structure and function of selected GPCR与酗酒相关的重构膜系统。 （i）探讨了GPCR视紫红质的表面是否应被视为均匀的，而周围的膜是连续的，或者是否应将特定的相互作用（尤其是与多不饱和脂质的特定相互作用）中的作用。通过1H MAS NMR研究了视紫红素与多饱和脂质的缔合，并从视紫红质转移到脂质。在牛杆外部段（ROS）磁盘以及含有牛Rhodopsin和脂质的重组膜上进行了实验，并具有多不饱和，docosahexaenoyl（DHA）和单不饱和烯烃链。在旋转的MAS转子中将视紫红素光活化，并随着光激活后的时间而磁化转移。所有Rhodopsin光中间体都优先转移了磁化强度到含DHA的脂质，但观察到元III Rhodopsin的最高速率。从蛋白质到DHA的磁化速率是脂质头组依赖性的，并且在序列PC，PS，PE中增加。在DHA含量的函数中进行的实验中，结果表明，多淡蛋白表面上的多饱和脂质与不同位点特异性相互作用。在毫秒或较短的时间尺度上，与脂质相关的脂质与脂质的大部分矩阵中的脂质快速交换。特定脂质 - 偶像蛋白关联的功能后果的解密需要进一步研究。 我们表征了与生物膜中所谓的筏子形成有关的液相相关的特性。尽管存在筏的生化证据很强，但事实证明，生物膜中筏的结构等效物的检测非常困难。在对特征良好的模型膜的实验中，表明液体有序结构域（LO）中脂质的1H MAS NMR光谱与液体无序（LD）和固体有序（SO）膜区具有明显不同的线宽。根据1H NMR偶极回声光谱的结果，LO相中的MAS共振扩大是由于相邻亚甲基之间的分子内质子质子偶极相互作用的有效强度的提高而导致的，这很可能是由于LO中Gauche/trans is构化的可能性较低。在记录的光谱中是温度的函数，LO结构域（RAFT）形成的开始被视为线条宽的突然发作。 LO相检测的1H MAS NMR方法不需要标记，并且可以应用于细胞膜中的筏子。关于生物膜的实验正在进行中。 （ii）我们开发了形成含有高浓度GPCR的管状单脂质双层的试剂和方法，这些双层含量覆盖了多孔阳极氧化铝（AAO）过滤器的孔壁。厚度为60微米的一个平方厘米的过滤材料可产生多达500 cm2的定向膜，这足以用于脂质基质的多核固态NMR研究，并在掺入的，同位素标记的蛋白质上。通过固态NMR方法，我们确定膜通过封闭且稳定的水垫与支撑分开。脂质小管的内表面可以从外部溶液中自由获取。基于氧化铝的支持为交换溶液，有效的颗粒保留，刚性，均匀的表面和透明度（湿时）提供了高流速的优势。从天然来源纯净的GPCR Rhodopsin，以及在大肠杆菌中表达的重组外周素大麻素受体CB2以功能形式纳入了管状双层。将牛Rhodopsin重构为AAO过滤器不会干扰视淡蛋白的功能。通过UV-VIS分光光度法监测的AAO孔中的Rhodopsin的光活化与无支撑的Unilamellar脂质体中的Rhodopsin无法区分。如A [35S] GTP-GAMMA-S结合测定法所示，AAO孔中的Rhodopsin具有G蛋白结合。通过2H NMR研究了脂质 - 偶像蛋白的相互作用在SN-1-或SN-2链中，或者SN-2链perdeperedErdeereRETERERERL-2-DOCOSAHEXAENOYL-SN-GLYCERO-3-甘油3-磷酸作为基质脂质。 Rhodopsin掺入增加了双层方向的镶嵌扩散，并在从纳米到微秒的范围内促进了运动的光谱密度，被发现是自旋旋转松弛时间的显着减少。由于与视紫红质相互作用而引起的脂质链顺序参数的变化微不足道。 （iii）在大肠杆菌中表达人类外周型大麻素受体（CB2），是与麦芽糖结合蛋白和硫氧还蛋白的融合。通过Western-印迹分析和质谱证实了全长融合的成功表达。通过各种天然和合成大麻素配体的广泛结合研究证实了受体在细菌原生质体膜中的功能活性和结构完整性。对表达重组CB2的尿素处理的大肠杆菌膜的激动剂刺激导致体外耦合测定中G蛋白的激活。为了纯化蛋白质，在多肽链中的各个位置引入了链球菌标签和dechistidine标签。 Fusion-CB2在TEV-蛋白酶裂解位点成功切割，而CB2通过亲和色谱的两个步骤纯化。纯化的CB2成功地重构为脂质双层，并至少部分保留了其配体结合特性。该方案允许通过NMR和散射技术表达和纯化重组受体的毫克重组受体。