NMR INVESTIGATIONS OF CELL MEMBRANE STRUCTURE

本项目的目标是：（1）研究酒精与生物膜中蛋白质和脂质的相互作用;（2）研究由具有多不饱和脂肪酸（如二十二碳六烯酸（DHA）22：6 n-3）的脂质组成的膜的结构和动力学;（3）研究与酒精中毒和脂质多不饱和相关的脂质-蛋白质相互作用。(1)我们得到了直接的证据，通过NMR表明，乙醇优先与膜的脂-水界面相互作用。乙醇相互作用由氢键和疏水相互作用的机会驱动。我们定量乙醇结合到膜组成的脂质和蛋白质在生理乙醇浓度为20 mM的顶空气相色谱法。这种方法非常适合分区研究，因为它是非扰动的。在生理条件下，约10%的总乙醇结合到脂质和蛋白质的界面上。NMR测量表明，游离和结合的乙醇分子是在快速交换，乙醇通过膜的速率仅略低于水的渗透率。乙醇的界面结合提高了表面的有效乙醇浓度，但降低了其在活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。在脂质膜中，这导致每个脂质分子的面积增加和脂质烃链的无序。乙醇诱导的链无序在多不饱和双层中较小，最有可能是因为多不饱和烃链已经占据了每个分子的较大面积，因此对乙醇诱导的无序不太敏感。在脂质-水界面处的乙醇分子阻断水通过脂质双层扩散的途径，如在水渗透速率降低中所见。(2)脑突触体和视网膜杆外节的膜含有30-50摩尔%的六重不饱和二十二碳六烯酸（DHA）作为脂烃链。DHA的一个可能作用是改变对受体蛋白活性重要的膜机械性质。有争议的性质的扰动，DHA链诱导膜烃秩序。DHA 22碳单元内的六个亚甲基中断的顺式双键减少了结构转变的自由度，这导致这些链具有特定的刚性构象如角铁或螺旋的建议。然而，直接测量DHA链序参数揭示了不同的情况。使用魔角旋转NMR实验，重新耦合13 C-1H偶极相互作用，指定的DHA的顺序参数，并通过X射线衍射确定的DHA链晶胞的尺寸。结果表明，膜中的DHA链更喜欢环状构象，并经历快速的结构转变，为富含受体的神经膜提供了更大的灵活性。我们开发了定量的方法来解释NMR NOESY交叉弛豫率之间的脂质共振。除了提供脂质结构的信息，这些速率是敏感的动态膜重组的相关时间范围从皮科到微秒。实验速率和速率从分子动力学计算的比较表明，由脂质分子的横向扩散引起的质子之间的距离变化是在脂质中的交叉弛豫的主要机制。该分析量化了生物膜中的高度分子紊乱，显示了即使是相邻脂质分子的最远区段（例如胆碱头基中的甲基和脂肪酸链的末端甲基）之间的接近的有限概率。分子间交叉弛豫速率是研究液晶相脂质横向结构的理想工具。可以检测不均匀的脂质分布和脂质物质相互作用的偏好，以及掺入膜中的物质的位置的偏好。(3)用固态~ 2 H NMR研究了HIV-1包膜糖蛋白gp 41羧基端的溶细胞肽片段828-848（P828）在膜中的行为。数据与N-末端肽区域部分渗透到膜的疏水核心中一致，而肽的C-末端部分保持在脂质/水界面附近。肽掺入的结果在一个显着减少的脂质链的顺序向双层中心，但只有适度减少附近的脂质甘油。此外，通过高分辨率NMR研究了肽在水中的游离结构和与SDS胶束结合的结构。P828在水中是非结构化的，但当与带负电荷的脂质体或胶束结合时以柔性的部分螺旋构象存在。柔性螺旋覆盖肽的前14个残基，而肽的C-末端似乎是非结构化的。肽诱导的脂质链有序分布的变化表明，膜曲率应力是P828细胞溶解行为的驱动力。

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.