NMR Investigations Of Cell Membrane Structure

该项目的目标是：（1）研究由含有多不饱和脂肪酸（例如二十二碳六烯酸（DHA）22：6 n-3）的脂质组成的膜的结构和动力学，（2）研究多不饱和脂质基质与G蛋白的相互作用偶联膜受体（GPCR）和（3）研究重组膜系统中与酒精中毒相关的选定GPCR的结构和功能。 (1)我们开发的方法，使用魔旋核磁共振与脉冲场梯度（PFG MAS NMR）的应用程序的研究脂质的横向扩散和域的形成在生物膜。这种方法测量从微米到纳米的长度尺度上的扩散，而不使用扰动标签。由魔角旋转提供的共振信号的优异分辨率允许对单个膜成分以及膜掺入的药物（例如G蛋白偶联膜受体的疏水配体）进行扩散测量。针对大量模型膜系统研究了侧向扩散。样品由纯化的脂质组成，比较了头基磷脂酰乙醇胺、磷脂酰丝氨酸和磷脂酰胆碱;比较了14、16和18个碳的链长;比较了链上沿着1至6个双键的不饱和度。高度不饱和的脂质，如那些与DHA烃链有显着较高的横向扩散率和较低的热活化能。我们将高扩散速率与我们最近报道的多不饱和链的惊人大的灵活性和快速的构象转变联系起来。基质脂质、水和膜溶解物质的横向扩散速率与膜组织有内在联系，包括域或筏的存在。结构域的形成进行了研究，在生物相关的磷酸二酰胆碱，磷脂酰乙醇胺和胆固醇的混合物。 (2)我们开发了用于在多孔固体支持物内形成含有高浓度膜受体的管状单脂质双层膜的试剂和方法。管状双层是用于生物传感器和结构研究的理想选择。一平方厘米的厚度为60微米的过滤材料可以产生高达500 cm 2的定向膜，这足以用于对脂质基质和掺入的同位素标记的蛋白质进行多核固态NMR研究。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物分离。脂质小管的内表面可从外部溶液自由进入。基于氧化铝的载体提供了交换溶液的高流速、有效的颗粒保留、刚性、均匀的表面和透明性（当湿润时）的优点。使用这种技术，G-蛋白偶联膜受体（GPCR）视紫红质，从天然来源纯化，以及重组外周大麻素受体，CB 2，在大肠杆菌中表达的功能形式纳入到管状双眼皮。该装置非常适合配体结合研究，包括药物测试。该技术可应用于多种膜受体，但似乎对GPCR特别有用。单脂双层的使用大大减少了配体与底物的非特异性相互作用，因此提高了结合研究的灵敏度和重现性。膜和固体支持物之间的水层防止受体功能的干扰。该底物与通过荧光、放射性示踪剂、NMR和其他方法的信号检测相容。考虑到制备这种系统的容易性，由多孔氧化铝过滤器支撑的含蛋白质的生物膜具有相当大的希望用于NMR结构研究以及生物传感器。利用饱和转移差谱结合魔角自旋技术成功地研究了含有二十二碳六烯酸的脂质分子与视紫红质的相互作用。结果表明，强烈的偏好与多不饱和二十二碳六烯酸的视紫红质的相互作用。 (3)人外周型大麻素受体（CB 2）在大肠杆菌中表达为与麦芽糖结合蛋白，硫氧还蛋白和十组氨酸标签的融合体。蛋白质印迹分析和质谱分析证实了全长融合蛋白的成功表达。通过与多种天然和合成大麻素配体的广泛结合研究证实了细菌原生质体膜中受体的功能活性和结构完整性。尿素处理的E.在体外偶联试验中，表达重组CB 2的大肠杆菌膜导致G蛋白的活化。通过固定化金属亲和层析，然后在洗涤剂的存在下通过离子交换层析将融合-CB 2蛋白纯化至85-90%。该方案允许表达和纯化毫克量的重组受体。通过特异性TEV蛋白酶的作用，可以从融合蛋白中除去N-和C-末端标签。通过对DPC胶束中的CB 2-融合的高分辨率NMR，确定纯化的CB 2与配体CP 55、940和花生四烯酸形成1：1复合物。CB 2融合成功地重组成磷脂酰胆碱双层和膜沉积到一个多孔基底作为管状脂质双层的结构研究，通过NMR和散射技术。

The objectives of this project are to: (1) study structure and dynamics of membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (2) study the interaction of the polyunsaturated lipid matrix with G-protein coupled membrane receptors (GPCR) and (3) investigate structure and function of selected GPCR with relevance for alcoholism in reconstituted membrane systems. (1) We developed methods to use magic spinning NMR with application of pulsed field gradients (PFG MAS NMR) for the study of lipid lateral diffusion and domain formation in biomembranes. This approach measures diffusion on the lengthscale from micro- to nanometers without the use of perturbing labels. The excellent resolution of resonance signals afforded by magic angle spinning permitted diffusion measurements for individual membrane constituents as well as for membrane incorporated drugs, e.g. the hydrophobic ligands of G-protein coupled membrane receptors. Lateral diffusion was studied for a large number of model membrane systems. The samples consisted of purified lipids comparing the headgroups phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine; contrasting chain lengths of fourteen, sixteen, and eighteen carbons; and comparing degrees of unsaturation from one to six double bonds along a chain. Highly unsaturated lipids like those with DHA hydrocarbon chains had significantly higher rates of lateral diffusion and lower thermal activation energies. We linked the high diffusion rates to the surprisingly large flexibility and the rapid conformational transitions of polyunsaturated chains which we had reported recently. The rates of lateral diffusion of matrix lipids, water, and membrane dissolved substances are intrinsically linked to membrane organization, including the presence of domains or rafts. Domain formation was studied in mixtures of biologically relevant phosphatdiylcholines, phosphatidylethanolamines, and cholesterol. (2) We developed reagents and methods for formation of tubular single lipid bilayer membranes containing high concentrations of membrane receptors inside porous solid supports. The tubular bilayers are ideal for use in biosensors and for structural studies. One square centimeter of the filter material with a thickness of 60 micrometers may yield 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). Using this technology, the G-protein coupled membrane receptors (GPCR) rhodopsin, purified from natural sources, as well as the recombinant peripheral cannabinoid receptor, CB2, expressed in E-coli were incorporated into the tubular bilaeyers in functional form. The setup is ideal for ligand binding studies, including drug testing. The technology may be applied to a broad variety of membrane receptors but appears to be particularly useful for GPCR. The use of single lipid bilayers greatly reduces nonspecific interactions of ligands with the substrate therefore enhancing sensitivity and reproducibility of binding studies. The water layer between the membrane and the solid support prevents perturbation of receptor function. The substrates are compatible with signal detection by fluorescence, radiotracers, NMR, and other methods. Considering the ease of preparation of such systems, protein containing biomembranes supported by the porous aluminum oxide filters have considerable promise for use in NMR structural studies as well as in biosensors. The tubular bilayers were used successfully to study the interaction of docosahexaenoic acid containing lipids with rhodopsin by saturation transfer difference NMR spectroscopy in combination with magic angle spinning. The results indicate a strong preference for interaction of rhodopsin with the polyunsaturated docosahexaenoic acid. (3) Human peripheral-type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose-binding protein, thioredoxin, and a decahistidine tag. 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 the urea-treated E. coli membranes expressing recombinant CB2 resulted in an activation of the G proteins in the in vitro coupled assay. The fusion-CB2 protein was purified to 85-90% by immobilized-metal affinity chromatography followed by ion-exchange chromatography in the presence of detergents. The protocol allows for expression and purification of milligram quantities of the recombinant receptor. N- and C-terminal tags can be removed from the fusion protein by action of a specific TEV protease. By high resolution NMR on the CB2-fusion in DPC micelles it was determined that purified CB2 forms 1:1 complexes with the ligands CP55,940 and anandamide. The CB2-fusion was successfully reconstituted into phosphatidylcholine bilayers and the membranes deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.