Nmr Investigations Of Cell Membrane Structure

细胞膜结构的核磁共振研究

• 批准号: 6508246
• 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/6508246
• 关键词: X ray crystallography; alcoholism /alcohol abuse; cell membrane; ethanol; intermolecular interaction; lipid bilayer membrane; lipid structure; membrane activity; membrane lipids; membrane proteins; membrane structure; nuclear magnetic resonance spectroscopy; omega 3 fatty acid; osmotic pressure; physical chemical interaction; protein structure; unsaturated fatty acids

The objectives of this project are to: (1) study structure and dynamics of membranes composed of lipids containing polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3, (2) study lipid-protein interactions related to lipid polyunsaturation and alcoholism, and (3) investigate the interaction of alcohol with proteins and lipids in biological membranes. (1) The membranes of brain synaptosomes and retinal rod outer segments contain 30-50 mol% of the six-fold unsaturated docosahexaenoic acid (DHA) as lipid hydrocarbon chains. One possible role of DHA is to alter membrane mechanical properties important for activity of receptor proteins. Using a magic angle spinning NMR experiment which re-couples 13C-1H dipolar interactions, assigned DHA order parameters were obtained. A unique membrane probe - perdeuterated DHA - was synthesized and incorporated into the lipid matrix. Twelve distinct order parameters were measured. Furthermore, the dimensions of the DHA chain unit cell were determined by x-ray diffraction. Order parameters of all methylene segments between double bonds in the hydrocarbon chain, and the order of the majority of double bonds is very low. Only the two methylene segments near the carboxyl group of DHA have order parameters that are comparable to values of more saturated chains. The low order is a reflection of both a change in bond geometry and an increase in chain motions. Experimental results were combined with results of simulations. The analysis suggests that DHA chains in membranes can exchange between looped, tilted, and extended conformations in rapid succession, providing increased flexibility to receptor-rich neural membranes. We developed quantitative methods for interpretation of NMR NOESY cross-relaxation rates between lipid resonances. In addition to providing information on lipid structure, these rates are sensitive to the dynamics of membrane reorganization in the correlation time range form pico- to microseconds. The comparison of experimental rates and rates from molecular dynamics simulations suggests that distance variation between protons caused by lateral diffusion of lipid molecules is the primary mechanism of cross-relaxation in lipids. The analysis quantifies the high degree of molecular disorder in biological membranes, showing a finite probability of close approach between even the most distant segments of neighboring lipid molecules (e.g. the methyl groups in the choline headgroup and the terminal methyl groups of the fatty acid chains). Intermolecular cross-relaxation rates are an ideal tool to study lateral lipid organization in the liquid-crystalline phase of lipids. Inhomogeneous lipid distribution and preferences in the interaction of lipid species, as well as preferences in the location of substances that incorporate into membranes can be detected. We developed approaches to conduct experiments on membrane samples oriented at solid interfaces and in lipid mixtures that orient spontaneously in the strong magnetic field of NMR instruments. The analysis of NMR lineshapes revealed the variable degree of mosaic spread in bilayer orientation for the different membranes. (2) There is evidence that a high content of DHA in retinal membranes modulates physical properties of membranes, creating an environment that is optimal for function of rhodopsin, the primary visual receptor, and a member of the G-protein coupled receptor family. We investigated this hypothesis by solid-state NMR methods. Rhodopsin was reconstituted into fully hydrated, solid-supported oriented multi-bilayer samples. Using 2H-labeled lipids, we compared lipid order parameters in the absence, and in the presence of the protein. We obtained highly resolved spectra from deuterated acyl chains in membranes containing a reconstituted integral membrane protein under physiological conditions. Oriented samples also improve NMR sensitivity enabling work with milligram-size samples. We have studied the phase diagram of the polyunsaturated 18:0-22:6 PE and demonstrated that it forms inverse hexagonal phases at all temperatures above the gel-fluid transition. The results suggest the existence of polyunsaturated lipid-induced membrane curvature stress that is likely to modulate the degree of activation of membrane incorporated receptors like rhodopsin. (3) Ethanol can act at multiple sites, with variable emphasis on interaction via the lipid matrix or via direct interaction with the protein, depending on the specific protein system involved. We propose that the binding of ethanol molecules to the lipid matrix of biomembranes is an important event in the action of ethanol on biological matter. We studied the interaction of ethanol with saturated, mono-, and polyunsaturated membranes quantitatively by MAS NOESY NMR. The resolution of resonance lines allows detection of 13-16 proton signals from lipid, ethanol, and water. Results of NMR measurements were combined with atomic-level molecular dynamics simulations to provide a deeper interpretation of experimental results. The site of ethanol interaction with the lipids is the primary factor that determines NMR cross-relaxation rates. Differences in correlation times and motional amplitudes of lipid segments play a secondary role. In particular, magnetization transfer to the headgroup choline resonance was somewhat lower than expected, due to fast lipid dynamics. As observed previously for lipid-lipid cross-relaxation, the rates scale with translational diffusion rates of ethanol in the bilayer. Ethanol resides mostly in the water phase, but binds for brief periods of time, of the order of nanoseconds, to the polar groups of the lipid/water interface, primarily to lipid phosphate groups. This temporary interaction introduces anisotropy into the motion of ethanol, with ethanol methylene C-D order parameters of 0.06. Cross-relaxation is strongest between ethanol and lipid resonances from the lipid/water interface including the glycerol, upper hydrocarbon chain, and lipid headgroup regions. There is evidence that the ethanol molecule in membranes is oriented preferentially with its methyl group toward the hydrophobic bilayer core. Cross-relaxation between ethanol and lipid hydrocarbon chains methyl is a reflection of both lipid hydrocarbon chain upturns and brief excursions of ethanol molecules into the upper region of lipid hydrocarbon chains. Overall, the probability of ethanol penetration into the center of the hydrophobic core of membranes is extremely low.

该项目的目的是：（1）由含有多不饱和脂肪酸的脂质组成的膜的研究结构和动态，例如二十二烷己己烯酸（DHA）22：6n-3，（2）研究脂质 - 蛋白质相互作用，与脂质多同性含量和酒精含量相关的蛋白质以及（3）与蛋白质相关的蛋白质和lip rip的含量。 （1）脑突触体和视网膜外部片段的膜包含30-50 mol％的六倍不饱和二十六烯酸（DHA）作为脂质烃链。 DHA的一种可能作用是改变对受体蛋白活性重要的膜机械性能。使用魔术角旋转NMR实验，该实验可以重新与13C-1H偶极相互作用，获得了分配的DHA顺序参数。合成了一个独特的膜探针 - perdeperated DHA-并掺入脂质基质中。测量了十二个不同的顺序参数。此外，通过X射线衍射确定DHA链晶胞的尺寸。烃链中双键和大多数双键的阶列段的顺序参数非常低。 DHA羧基附近的两个亚甲基段具有与更饱和链值相当的顺序参数。低阶是键几何形状变化和链运动增加的反映。实验结果与模拟结果结合使用。分析表明，膜中的DHA链可以快速连续的循环，倾斜和扩展构象之间交换，从而提高了对富含受体的神经膜的灵活性。我们开发了用于解释脂质共振之间NMR NOESY交叉解释率的定量方法。除了提供有关脂质结构的信息外，这些速率还对相关时间范围内的膜重组的动力学敏感。分子动力学模拟实验速率和速率的比较表明，脂质分子的横向扩散引起的质子之间的距离变化是脂质交叉浮肿的主要机制。该分析量化了生物膜中的高度分子障碍，显示了甚至在相邻脂质分子的最遥远段之间接近接近的有限概率（例如，胆碱头组中的甲基和脂肪酸链的末端甲基甲基甲基）。分子间交叉解释率是研究脂质液晶期侧脂质组织的理想工具。可以检测到脂质物种相互作用的不均匀脂质分布和偏好，以及在掺入膜中的物质位置的偏好。我们开发了对在固体界面和脂质混合物中定向的膜样品上进行实验的方法，这些脂质混合物在NMR仪器的强磁场中自发定向。 NMR线形的分析揭示了不同膜的双层方向中镶嵌物扩散程度的变化程度。 （2）有证据表明，视网膜膜中DHA的高含量调节了膜的物理性质，创造了一种对Rhodopsin，主要视觉受体的功能以及G蛋白耦合受体家族的成员最佳的环境。我们通过固态NMR方法研究了这一假设。将视紫红质重构为完全水合的固体支持的多双层样品。使用2H标记的脂质，我们在不存在的情况下和存在蛋白质的情况下比较了脂质阶参数。我们从生理条件下的膜膜中从含有重构积分膜蛋白的膜中获得了高度分辨的光谱。面向的样品还提高了NMR敏感性，从而可以使用毫克大小的样品进行工作。我们已经研究了多不饱和的18：0-22：6 PE的相图，并证明它在凝胶 - 富流晶过渡上方的所有温度下形成了六角形相。结果表明存在多不饱和脂质诱导的膜曲率应激，这可能会调节膜掺入的受体（如视紫红质）的激活程度。 （3）乙醇可以在多个位点起作用，其强调通过脂质基质或通过与蛋白质直接相互作用的相互作用，具体取决于所涉及的特定蛋白质系统。我们建议，乙醇分子与生物膜的脂质基质的结合是乙醇对生物学物质作用的重要事件。我们通过MAS Noesy NMR定量地研究了乙醇与饱和，单和多不饱和膜的相互作用。共振线的分辨率允许检测脂质，乙醇和水的13-16质子信号。 NMR测量结果与原子级分子动力学模拟相结合，以更深入地解释实验结果。乙醇与脂质的相互作用位点是决定NMR交叉解释率的主要因素。脂质段的相关时间和运动幅度的差异起次要作用。特别是，由于快速脂质动力学，磁化强度转移到头基胆碱共振略低于预期。如先前观察到的脂质脂质交叉解释，速率尺度的速度尺度随双层中乙醇的平移速率。 乙醇主要存在于水期，但纳米秒的顺序与脂质/水界面的极性组结合，主要与脂质磷酸盐基团结合。这种暂时的相互作用将各向异性引入乙醇的运动，乙醇甲醇C-D阶参数为0.06。乙醇和脂质共振之间的交叉浮肿最强，包括甘油，上碳氢化合物链和脂质头组区域。有证据表明，膜中的乙醇分子优先定向其甲基朝向疏水双层核心。乙醇和脂质烃之间的跨浮雕甲基甲基是脂质烃链上的反映，并将乙醇分子的短暂偏移到脂质碳氢化合物链的上区域。总体而言，乙醇渗透到膜核心核心中心的可能性极低。