NMR Investigations of Cell Membrane Structure

细胞膜结构的核磁共振研究

• 批准号: 6818422
• 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/6818422
• 关键词: G protein; alcoholism /alcohol abuse; bioimaging /biomedical imaging; cell membrane; cell surface receptors; conformation; ethanol; intermolecular interaction; lipid structure; membrane activity; membrane lipids; membrane permeability; membrane proteins; membrane reconstitution /synthesis; membrane structure; molecular assembly /self assembly; nuclear magnetic resonance spectroscopy; omega 3 fatty acid; osmotic pressure; physical chemical interaction; protein structure; receptor coupling; structural biology; unsaturated fatty acids

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.

该项目的目标是：（1）由脂质与多不饱和脂肪酸组成的膜的研究结构和动态，例如二十二乙烯二烯酸（DHA）22：6n-3，（2）研究与脂质脂质成分与GPORETIN辅助偶联膜GPCR的相关结构和（3）研究的相互作用（2）在重组的膜系统中酒精中毒。 （1）我们最近的实验表明，多不饱和脂肪酸（PUFA）是在具有快速构象转变的多种构象中存在的高度柔性分子。甘油基团附近的PUFA的移动性与其他链相似。但是，相关时间减少，运动幅度从双键到双键增加，在PUFA链的甲基末端达到了10 ps的相关时间。尽管顺式锁定的双键具有刚性的刚度，PUFA的动作速度更快，运动幅度较大。这种灵活性的根本原因是双键之间C-C键旋转的潜在障碍。低电位允许PUFA迅速改变构象，而无需大量的惩罚。我们已经检测到Omega-3 docosahecahexaenoic Acid（22：6N3，DHA）和沿Bilayer正常的Omega-6 docosapentaenoic酸（22：5N6，DPA）之间的PUFA链密度分布显着差异。与DPA相比，与DPA相比，DHA往往具有更高的密度，这是从链顺序参数的差异中得出的，在沿链条的每个碳原子的分辨率下，在链条的每个碳原子的分辨率下，通过X射线衍射实验获得的脂质双层的电子密度曲线（与Dr. tristram-nagle-nagle）和模拟（与Dr. tristram-nagle）和simulaction（ander）和simculation（and）和simals and the the n of the and the and the and the and ins of the-nagle）和。我们的观察结果清楚地表明，富含DHA或DPA的膜之间的生物物理特性差异。我们推测，脂质烃链分布的差异改变了膜的侧向压力密度曲线，这改变了GPCR在配体结合时激活的概率。 （2）视紫红质和其他膜蛋白控制其局部环境的脂质组成的机制，例如通过脂质筏的形成，引起了很大的关注。我们正在开发魔术角旋转（MAS）NMR方法，以表征膜成分的侧向分布。通过MAS NMR同时应用脉冲现场梯度，一种新型的实验方法，我们测量了脂质和膜相关物质的横向扩散速率，例如药物或GPCR的内源性配体，例如。多不饱和的anandamide。新型的MAS NMR技术也用于研究多不饱和脂质和GPCR之间的特定相互作用。与饱和链相比，我们合作者对含有牛Rhodopsin的重构膜以及分子模拟的实验表明，DHA链更深地穿透到GPCR的跨膜区域。 （3）工作已经开始表达GPCR作为融合蛋白，用于重构膜的功能和结构研究。