NMR INVESTIGATIONS OF CELL MEMBRANE STRUCTURE

细胞膜结构的核磁共振研究

• 批准号: 6431350
• 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/6431350
• 关键词: X ray crystallography; alcoholism /alcohol abuse; cell membrane; ethanol; lipid bilayer membrane; lipid structure; membrane lipids; membrane proteins; membrane structure; nuclear magnetic resonance spectroscopy; osmotic pressure; physical chemical interaction; protein structure; unsaturated fatty acids

The objectives of this project are to: (1) investigate the interaction of alcohol with proteins and lipids in biological membranes; (2) study structure and dynamics of membranes composed of lipids with polyunsaturated fatty acids such as docosahexaenoic acid (DHA) 22:6n-3; and (3) study lipid-protein interactions related to alcoholism and lipid polyunsaturation. (1) We obtained direct evidence by NMR that ethanol interacts preferentially with the lipid-water interface of membranes. Ethanols interactions are driven by both the opportunity for hydrogen bonding and hydrophobic interactions. We quantitated ethanol binding to membranes composed of lipids and proteins at the physiological ethanol concentration of 20 mM by headspace gas chromatography. This method is ideally suited for partitioning studies because it is non-perturbing. Under physiological conditions, of the order of 10% of total ethanol is bound to the interfaces of lipids and proteins. NMR measurements indicate that free and bound ethanol molecules are in rapid exchange, and that ethanol passes through membranes at rates that are only slightly lower than permeation rates of water. Interfacial binding of ethanol raises effective ethanol concentrations at surfaces but lowers its concentration in the electrolyte solutions of living organisms. The interface location of ethanol lowers interfacial energy of lipids and proteins. In lipid membranes this results in an increase of area per lipid molecule and a disordering of lipid hydrocarbon chains. Ethanol-induced chain disordering is smaller in polyunsaturated bilayers, most likely because polyunsaturated hydrocarbon chains already occupy a larger area per molecule and are therefore less sensitive to ethanol-induced disordering. The ethanol molecules at the lipid-water interface block pathways for water diffusion through lipid bilayers as seen in decreased rates of water permeation. (2) 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. There is controversy as to the nature of the perturbation which DHA chains induce on membrane hydrocarbon order. The six methylene-interrupted cis double bonds within DHAs 22 carbon unit reduce the number of degrees of freedom for structural transitions, which led to the suggestion that these chains have a specific rigid conformation such as angle-iron or helical. However, direct measurements of DHA chain order parameters reveal a different picture. Using a magic angle spinning NMR experiment which re-couples 13C-1H dipolar interactions, assigned DHA order parameters were obtained, and dimensions of the DHA chain unit cell were determined by x-ray diffraction. The results suggest that DHA chains in membranes prefer looped conformations and undergo rapid structural transitions, 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 calculations 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. (3) The behavior of the cytolytic peptide fragment 828-848 (P828) from the carboxy-terminus of the envelope glycoprotein gp41 of HIV-1 in membranes was investigated by solid state 2H NMR on P828 with the selectively deuterated isoleucines I3, I13, I16, and I20. The data are consistent with partial penetration of the N-terminal peptide region into the hydrophobic core of the membrane, while the C-terminal portion of the peptide remains near the lipid/water interface. Peptide incorporation results in a significant reduction of lipid chain order toward the bilayer center, but only a modest reduction near the lipid glycerol. In addition, the structure of the peptide was investigated free in water and bound to SDS micelles by high resolution NMR. P828 is unstructured in water but exists in a flexible, partially helical conformation when bound to negatively charged liposomes or micelles. The flexible helix covers the first 14 residues of the peptide, whereas the C-terminus of the peptide appears to be unstructured. The peptide-induced changes in lipid chain order profiles indicate that membrane curvature stress is the driving force for the cytolytic behavior of P828.

该项目的目标是：（1）研究酒精与蛋白质和脂质在生物膜中的相互作用； （2）由脂质组成的膜的研究结构和动力学，具有多不饱和脂肪酸，例如二十六烯酸（DHA）22：6n-3； （3）研究与酒精中毒和脂质多不饱和有关的脂质蛋白相互作用。 （1）我们通过NMR获得了直接证据，表明乙醇与膜的脂质 - 水界面优先相互作用。乙醇相互作用是由氢键和疏水相互作用的机会驱动的。我们通过顶空气相色谱法定量了在20 mM的生理乙醇浓度下定量由脂质和蛋白质组成的乙醇的结合。该方法理想地适合分区研究，因为它是不受干扰的。在生理条件下，总乙醇的10％的阶与脂质和蛋白质的界面结合。 NMR测量表明，游离和结合的乙醇分子正在快速交换，并且乙醇以仅略低于水的渗透率的速率通过膜。乙醇的界面结合在表面上提高了有效的乙醇浓度，但降低了其在生物体的电解质溶液中的浓度。乙醇的界面位置降低了脂质和蛋白质的界面能量。在脂质膜中，这会导致每个脂质分子面积增加和脂质烃链的无序。乙醇诱导的链错无序在多不饱和双层中较小，很可能是因为多不饱和烃链已经占据每个分子的面积较大，因此对乙醇诱导的无序无序敏感。脂质 - 水界面处的乙醇分子通过脂质双层扩散的水扩散，如水渗透速率降低所示。 （2）脑突触体和视网膜外部片段的膜包含30-50 mol％的六倍不饱和二十二烯酸（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。关于DHA链在膜碳氢化合物顺序诱导的扰动的性质存在争议。 DHAS 22碳单元内的六种甲基二键式顺式双键减少了结构过渡的自由度的数量，这导致了这些链具有特定的刚性构象（例如Angle-riron或Helical）的建议。但是，DHA链顺序参数的直接测量显示出不同的图像。使用魔术角旋转NMR实验，可以获得重新与13C-1H偶极相互作用，分配的DHA顺序参数，并通过X射线衍射来确定DHA链晶胞单元的尺寸。结果表明，膜中的DHA链更喜欢循环构象和快速的结构过渡，从而增加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。实验率和分子动力学计算速率的比较表明，脂质分子横向扩散引起的质子之间的距离变化是脂质中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。 (3) The behavior of the cytolytic peptide fragment 828-848 (P828) from the carboxy-terminus of the envelope glycoprotein gp41 of HIV-1 in membranes was investigated by solid state 2H NMR on P828 with the selectively deuterated isoleucines I3, I13, I16, and I20.数据与N末端肽区域的部分穿透到膜的疏水核心，而肽的C末端部分保持在脂质/水界面附近。肽掺入导致脂质链阶向双层中心显着降低，但在脂质甘油附近只有适度的还原。此外，研究肽的结构在水中游离并通过高分辨率NMR与SDS胶束结合。 p828在水中无组织，但与带负电荷的脂质体或胶束结合时，存在于柔性的，部分螺旋的构象中。柔性螺旋覆盖了肽的前14个残基，而肽的C末端似乎是非结构化的。肽诱导的脂质链顺序变化表明，膜曲率应力是p828的细胞溶解行为的驱动力。