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)我们最近的实验表明，多不饱和脂肪酸(PUFA)是一种高度灵活的分子，存在于多种构象中，具有快速的构象转变。多不饱和脂肪酸在甘油基团附近的迁移率与其他链相似。但在PUFA链的甲基末端，关联次数减少，运动幅度从双键到双键增加，达到10ps量级的关联时间。尽管顺式双键具有刚性，但多不饱和脂肪酸具有更快的运动速度和更大的运动幅度。这种灵活性的根本原因是围绕双键之间的C-C键旋转的极低势垒。低电势允许多不饱和脂肪酸快速改变构象，而不会产生明显的能量损失。我们发现omega-3二十二碳六烯酸(22：6n3，DHA)和omega-6二十二碳五烯酸(22：5n6，DPA)在双分子层上的链密度分布有显著差异。与DPA相比，DHA在脂水界面附近往往具有更高的密度，这是由于链有序参数、链上每个碳原子的运动关联时间与分辨率、通过X射线衍射实验(与Tristram-Nagle博士合作)和模拟(与Feller博士合作)获得的脂质双分子层的电子密度分布的不同而得出的。我们的观察清楚地指出，富含DHA或DPA的膜之间的生物物理性质存在差异。我们推测，脂族碳氢链分布的差异改变了膜的侧向压力密度分布，从而改变了GPCR在配体结合时被激活的可能性。 (2)视紫红质和其他膜蛋白控制其局部环境的脂组成的机制，例如通过形成脂筏，引起了人们的极大关注。我们正在开发魔角旋转(MAS)核磁共振方法来表征膜组分的横向分布。通过同时施加脉冲梯度的MAS核磁共振，我们测量了脂质和膜相关物质(如药物或GPCR的内源性配体，如多不饱和氨基甲酰胺)的侧向扩散速率。新的MAS核磁共振技术也被应用于研究多不饱和脂类与gpr之间的特异性相互作用。对含有牛视紫质的重组膜的实验以及我们合作者的分子模拟表明，与饱和链相比，DHA链更深入地渗透到GPCR的跨膜区。 (3)重组膜功能和结构研究的融合蛋白GPCR3已经开始表达。

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) Our recent experiments indicate that polyunsaturated fatty acids (PUFA) are highly flexible molecules existing in a multitude of conformations with rapid conformational transitions. The mobility of PUFA near the glycerol group is similar to other chains. However, the correlation times decrease and the motional amplitudes increase from double bond to double bond, reaching correlation times of the order of 10 ps at the methyl terminal end of the PUFA chain. Despite the rigidity of the cis-locked double bonds PUFA have faster motions and larger motional amplitudes. The underlying cause for this flexibility is an extremely low potential barrier for rotations about the C-C bonds between the double bonds. The low potentials permit PUFA to rapidly change conformation without significant energetic penalty. We have detected significant differences in the distribution of PUFA chain density between the omega-3 docosahexaenoic acid (22:6n3, DHA) and the omega-6 docosapentaenoic acid (22:5n6, DPA) along the bilayer normal. The DHA tends to have higher density near the lipid water interface compared to the DPA as derived from differences in chain order parameters, in motional correlation times with resolution for every carbon atom along the chain, in the electron density profiles of lipid bilayers obtained by x-ray diffraction experiments (collaboration with Dr. Tristram-Nagle), and in the simulations (collaboration with Dr. Feller). Our observations clearly point toward a difference in biophysical properties between membranes rich in DHA or DPA. We speculate that the differences in the distribution of lipid hydrocarbon chains alter lateral pressure density profiles of membranes which alter the probability of GPCR to activate upon ligand binding. (2) The mechanism by which rhodopsin and other membrane proteins control the lipid composition of their local environments, e.g. through the formation of lipid rafts, has attracted considerable attention. We are developing magic angle spinning (MAS) NMR approaches to characterize lateral distribution of membrane constituents. By MAS NMR with simultaneous application of pulsed field gradients, a novel experimental approach, we measured rates of lateral diffusion of lipids and membrane associated substances such as drugs or endogenous ligands of GPCR, e.g. the polyunsaturated anandamide. The novel MAS NMR techniques are also applied to study specific interactions between polyunsaturated lipids and GPCR. Experiments on reconstituted membranes containing bovine rhodopsin as well as molecular simulations by our collaborators suggest a deeper penetration of DHA chains into the transmembrane region of the GPCR compared to saturated chains. (3) Work has begun to express GPCR as fusion proteins for functional and structural studies on reconstituted membranes.