Nmr Investigations Of Cell Membrane Structure

该项目的目的是：(1)研究含有多不饱和脂肪酸（如二十二碳六烯酸（DHA） 22:6n-3）的脂质组成的膜的结构和动力学，(2)研究脂质多不饱和和酒精中毒相关的脂质-蛋白相互作用，(3)研究酒精与生物膜中蛋白质和脂质的相互作用。(1)脑突触体膜和视网膜杆外节含有30-50 mol%的六倍不饱和二十二碳六烯酸（DHA）作为脂质烃链。DHA的一个可能作用是改变对受体蛋白活性很重要的膜力学特性。采用魔角自旋核磁共振实验对13C-1H偶极相互作用进行重偶联，得到了分配的DHA序参量。合成了一种独特的膜探针-渗透DHA -并将其掺入脂质基质中。测量了12个不同的顺序参数。此外，通过x射线衍射测定了DHA链单元胞的尺寸。烃链中所有亚甲基段的双键之间的序参量，大部分双键的序参量都很低。只有靠近DHA羧基的两个亚甲基片段的顺序参数与更饱和的链的值相当。低阶反应了键几何形状的变化和链运动的增加。实验结果与仿真结果相结合。分析表明，膜中的DHA链可以在环状、倾斜和延伸构象之间快速连续交换，为富含受体的神经膜提供了更大的灵活性。我们开发了定量方法来解释脂质共振之间的NMR noasy交叉松弛率。除了提供有关脂质结构的信息外，这些速率在相关时间范围内（从皮秒到微秒）对膜重组的动态很敏感。实验速率和分子动力学模拟速率的比较表明，脂质分子横向扩散引起的质子间距离变化是脂质交叉弛豫的主要机制。该分析量化了生物膜中高度的分子紊乱，显示出即使是邻近脂质分子的最远段（例如胆碱头基的甲基和脂肪酸链的末端甲基）之间也有有限的接近概率。分子间交叉弛豫率是研究脂质液晶相中脂质横向组织的理想工具。可以检测到脂质分布的不均匀性和脂质相互作用中的偏好，以及纳入膜的物质的位置偏好。我们开发了在固体界面和脂质混合物中进行膜样品定向实验的方法，这些膜样品在核磁共振仪器的强磁场中自发定向。核磁共振谱分析揭示了不同膜在双层取向上的不同程度的镶嵌扩散。(2)有证据表明，视网膜膜中高含量的DHA可以调节膜的物理特性，为视紫红质（主要视觉受体）和g蛋白偶联受体家族成员的功能创造最佳环境。我们用固态核磁共振方法研究了这一假设。将视紫红质重构成完全水合的、固体支撑的定向多层样品。使用2h标记的脂质，我们比较了在没有和有蛋白质的情况下脂质顺序参数。我们在生理条件下获得了含有重组整体膜蛋白的膜中氘化酰基链的高分辨光谱。定向样品也提高了核磁共振灵敏度，使工作与毫克大小的样品。我们研究了多不饱和聚乙烯18:0-22:6的相图，证明了它在凝胶-流体转变以上的所有温度下都形成逆六方相。结果表明，多不饱和脂质诱导的膜曲率应力的存在可能调节视紫红质等膜结合受体的激活程度。(3)乙醇可以在多个位点起作用，通过脂质基质或通过直接与蛋白质相互作用的重点不同，这取决于所涉及的特定蛋白质系统。我们认为乙醇分子与生物膜脂质基质的结合是乙醇作用于生物物质中的一个重要事件。我们研究了乙醇与饱和膜、单不饱和膜和多不饱和膜的相互作用。共振线的分辨率允许从脂质、乙醇和水检测13-16个质子信号。核磁共振测量结果与原子水平的分子动力学模拟相结合，为实验结果提供了更深层次的解释。乙醇与脂质相互作用的位置是决定核磁共振交叉弛豫速率的主要因素。脂质段相关时间和波动幅度的差异起次要作用。特别是，由于快速的脂质动力学，磁化转移到头基团胆碱共振比预期的要低一些。正如先前观察到的脂质交叉松弛，速率与乙醇在双分子层中的平移扩散速率成比例。乙醇主要存在于水相，但能与脂/水界面的极性基团（主要是脂质磷酸基）结合很短的时间，大约在纳秒量级。这种临时相互作用使乙醇的运动具有各向异性，乙醇亚甲基C-D阶参数为0.06。乙醇和脂质之间的交叉松弛是最强的，这些共振来自脂质/水界面，包括甘油、上碳氢链和脂质头基区域。有证据表明，膜中的乙醇分子优先取向，其甲基朝向疏水双分子层核心。乙醇和脂质烃链甲基间的交叉弛豫反映了脂质烃链的上移和乙醇分子进入脂质烃链上部区域的短暂偏移。总的来说，乙醇渗透到疏水膜核心中心的可能性极低。

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.