NMR Investigations Of Cell Membrane Structure

本项目的目的是：(1)研究脂类与二十二碳六烯酸(DHA)22：6N-3等多不饱和脂肪酸组成的膜的结构和动力学；(2)研究多不饱和脂基质与G蛋白偶联膜受体(GPCR)的相互作用；(3)研究重组膜系统中与酒精中毒相关的GPCRs的结构和功能。 (1)发展了利用脉冲磁场梯度的魔旋核磁共振(PFG-MAS)研究生物膜中脂质横向扩散的方法。这种方法在微米长度尺度上测量扩散，而不使用扰动标签。魔角旋转提供的共振信号的出色分辨率允许对单个膜组分以及膜结合药物(例如G蛋白偶联膜受体的疏水配体)进行扩散测量。对大量的模型膜系统进行了横向扩散研究。样品由纯化的脂类组成，比较了头基磷脂酰乙醇胺、磷脂酰丝氨酸和磷脂酰胆碱；对比了14个、16个和18个碳的链长；以及比较了沿着链的1到6个双键的不饱和程度。具有DHA碳氢链的高不饱和脂类具有显著较高的横向扩散速率和较低的热激活能。我们将高扩散速率与我们最近报道的多不饱和链惊人的大灵活性和快速构象转变联系在一起。基质脂类、水和膜溶解物质的横向扩散速率与膜的组织结构有内在联系，包括结构域或木筏的存在。在与生物相关的磷脂酰胆碱、磷脂酰乙醇胺和胆固醇的混合物中研究了结构域的形成。 (2)我们开发了在多孔固体载体中形成含有高浓度膜受体的管状单脂双层膜的试剂和方法。管状双层膜非常适合用于生物传感器和结构研究。1平方厘米的厚度为60微米的过滤材料可以产生高达500 cm2的取向膜，这足以用于对脂质基质和掺入的同位素标记蛋白质的多核固体核磁共振研究。通过固体核磁共振方法，我们确定了膜与载体之间是由一个封闭而稳定的水垫分离的。脂质小管的内表面可以从外部溶液自由接触到。基于氧化铝的支撑体具有以下优点：交换溶液的流速高、有效的颗粒保持、坚硬、均匀的表面和透明(湿时)。利用这一技术，从天然来源纯化的G蛋白偶联膜受体(GPCR)以及在大肠杆菌中表达的重组受体以功能形式被整合到双层中。该装置非常适合进行配体结合研究，包括药物测试。这项技术可以应用于多种膜受体，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，从而提高了结合研究的灵敏度和重复性。膜和固体载体之间的水层防止了受体功能的干扰。该底物与荧光、放射性示踪剂、核磁共振和其他方法的信号检测兼容。考虑到这类体系的制备容易，由多孔氧化铝过滤器支撑的含有蛋白质的生物膜在核磁共振结构研究和生物传感器中有很大的应用前景。 (3)人外周型大麻素受体(CB2)在大肠杆菌中以麦芽糖结合蛋白(N端)和硫氧还蛋白(C端)与10个组氨酸残基融合的形式表达。Western-blotting分析证实全长融合基因成功表达。表达的受体定位于大肠杆菌细胞的内膜部分。融合蛋白经固定化金属亲和层析和离子交换层析后，纯度可达85-90%。该方案允许表达和纯化毫克量的重组受体。N-末端和C-末端标签可以通过特定的TEV蛋白酶的作用从融合蛋白中移除。通过与人工合成的大麻素配体的滤膜结合实验，证实了在大肠杆菌中表达的CB2的功能活性。在体外偶联实验中，用激动剂刺激表达重组CB2的大肠杆菌膜后，G蛋白被激活。初步结果表明，重组受体有可能成功地重组到脂质基质中，并沉积到适合随后通过核磁共振光谱和中子散射技术进行结构表征的固体载体上。

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 in biomembranes. This approach measures diffusion on the micrometer lengthscale without the use of perturbing labels. The excellent resolution of resonance signals afforded by magic angle spinning permited 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, G-protein coupled membrane receptors (GPCR), purified from natural sources, as well as recombinant receptors expressed in E-coli were incorporated into the bilayer 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. (3) Human peripheral-type cannabinoid receptor (CB2) was expressed iin E. colii as a fusion with maltose-binding protein (at the N-terminal end) and thioredoxin followed by ten histidine residues (at the C-terminal end). Successful expression of the full-length fusion was confirmed by Western-blot analysis. The expressed receptor was localized in the inner-membrane fraction of E. coli cells. 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. The functional activity of CB2 expressed in E. coli was confirmed by filter-binding assays with 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. Preliminary results suggest the possibility of successful reconstitution of the recombinant receptor into a lipid matrix, and deposition onto a solid support suitable for subsequent structural characterization by nuclear magnetic resonance spectroscopy and neutron scattering techniques.