NMR Investigations Of Cell Membrane Structure

细胞膜结构的核磁共振研究

• 批准号: 8148170
• 负责人: KLAUS GAWRISCH
• 金额: $ 198.98万
• 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/8148170
• 关键词: Cell Membrane Structures; Investigation

(i) The effect of membrane composition on ethanol partitioning into lipid bilayers was assessed by headspace gas chromatography. A series of model membranes with different compositions have been investigated. Membranes were exposed to a physiological ethanol concentration of 20 mM. The concentration of membranes was 20 wt% which roughly corresponds to values found in tissue. Partitioning depended on the chemical nature of polar groups at the lipid-water interface. Compared to phosphatidylcholine, lipids with headgroups containing phosphatidylglycerol, phosphatidylserine, and sphingomyelin showed enhanced partitioning while headgroups containing phosphatidylethanolamine resulted in a lower partition coefficient. The molar partition coefficient was independent of a membranes hydrophobic volume. This observation is in agreement with our previously published NMR results which showed that ethanol resides almost exclusively within the membrane-water interface. At an ethanol concentration of 20 mM in water, ethanol concentrations at the lipid/water interface are in the range from 30 - 15 mM, corresponding to one ethanol molecule per 100-250 lipids. We obtained evidence for critical behavior in cholesterol-rich model membranes that form coexisting liquid ordered and disordered phases which have been linked to raft formation in biological membranes. Deuterium NMR was used to evaluate phase boundaries in cholesterol containing ternary lipid membranes. The precise thermodynamic description of phase behavior permitted to predict composition and temperature at which critical behavior occurs. NMR resonances are dramatically broadened in the vicinity of critical points confirming their existence. Broaden-ing was attributed to increased spin-spin relaxation rates arising from modulations of chain order on a microsecond timescale. We speculate that spectral broadening is a reflection of formation of lipid-cholesterol clusters with microsecond lifetimes. Critical fluctuations provide a mechanism to produce lipidic structures with submicron dimensions at physiologically relevant composition and temperatures. Work on this project has been a collaborative research effort between Dr. Sarah Veatch, Dr. Sarah Keller, and the NMR Section of LMBB. In the framework of this project we developed NMR tools for detection of ordered lipid domains in biological membranes that do not require isotopic labeling. In collaboration with Dr. Joshua Zimmerbergs laboratory at NIH, those tools have been used to search for ordered lipid domains in intact influenza virus. Evidence for coexistence of ordered and disordered lipid domains in both the intact virus envelope and in bilayers made from extracted viral lipid was obtained. The functional implications of formation of ordered lipid domains in viral membranes are under investigation. (ii) We considered the issue of whether or not shifts in the metarhodopsin I (MI) metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area per lipid molecule and the membrane hydrophobic thickness were derived from measurements of the 2H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain. We explored if the surface of the GPCR rhodopsin should be viewed as homogeneous and the surrounding membrane as a continuum, or if specific interactions, in particular with polyunsaturated lipids may play a role in rhodopsin activation. The association of rhodopsin with poly- and monounsaturated lipids was studied by 1H MAS NMR with magnetization transfer from rhodopsin to lipid. It was shown that poly- and monounsaturated lipids interact specifically with different sites on rhodopsin. Associated lipids are in fast exchange with lipids of the matrix on a time-scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids suggesting stronger interactions. Highest rates were observed for Meta-III rhodopsin. Interactions with DHA-containing lipids are headgroup dependent and strength increased in the sequence phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine. We used 1H MAS NMR on rhodopsin in intact rod outer segment disks to track changes in rhodopsin hydration upon photoactivation. The data indicate that metarhodopsin-I differs from dark adapted rhodopsin and other photointermediates by a higher level of hydration. Although these experiments do not directly determine where on the protein hydration changes, magnetization transfer is most efficient when the interactions are both short-range (< 5 ) and long-lived (1-10 ns), conditions best satisfied by hydration of the protein core. Changes of GPCR hydration upon activation make GPCR signaling particularly sensitive to environmental factors that influence activity of water molecules. The same technique was applied to study hydration of membrane-embedded voltage-sensing protein domains in collaboration with the laboratory of Kenton Swartz, NINDS. (iii)Cannabinoid receptors have attracted much attention because of their role in health and disease including alcoholism. The peripheral cannabinoid receptor, CB2, is involved in immune and hormonal response. CB2 was expressed in Escherichia coli as a fusion with maltose-binding protein and several affinity tags. The fusion was cleaved and the receptor purified by Ni-NTA and Streptactin affinity chromatography in the presence of detergents. Several methods for reconstitution of CB2 into lipid bilayers (rapid dilution, dialysis, removal of detergents with absorbing resins) were compared. Composition, size, and homogeneity of proteoliposomes were investigated by analytical NMR, fluorescence spectroscopy using labeled lipid and CB2, dynamic light scattering, and sucrose gradient centrifugation. The protein was successfully stabilized during purification and reconstitution by a proper mixture of detergents, lipids, as well as ligand. Preparation of samples that contain milligrams of 90% functional CB2 was successful. The structural integrity of the reconstituted protein was confirmed by its ability to activate G-protein in response to agonist binding. We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The fermentation process produced about 2 mg of purified, labeled and functional CB2 per liter of culture medium.

（i）通过顶空气相色谱法评估膜组成对将乙醇分配到脂质双层中的影响。已经研究了一系列具有不同组成的模型膜。膜暴露于20 mm的生理乙醇浓度。膜的浓度为20 wt％，大致对应于组织中的值。分区取决于脂质 - 水界面上极性基团的化学性质。与磷脂酰胆碱相比，含有含磷脂酰甘油，磷脂酰甲酯和鞘磷脂的头组的脂质显示出增强的分配，而含有磷脂酰乙醇胺的头组则导致较低的分区系数。摩尔分区系数独立于疏水膜。该观察结果与我们先前发表的NMR结果一致，该结果表明乙醇几乎完全存在于膜 - 水界面中。在水中20 mm的乙醇浓度下，脂质/水界面处的乙醇浓度在30-15 mm的范围内，对应于每100-250个脂质的一个乙醇分子。 我们获得了富含胆固醇的模型膜中关键行为的证据，这些膜形成了并存的液相有序和无序相，这些相位与生物膜中的筏形成有关。 NMR氘用于评估含有三元脂质膜的胆固醇中的相边界。允许预测发生临界行为的组成和温度的相行为的精确热力学描述。 NMR共鸣在确认其存在的关键点附近得到了极大的扩展。扩展归因于自旋旋转弛豫率提高，这是由于微秒时间尺度上的链顺序调制而产生的。我们推测光谱扩大是用微秒生命的脂质 - 胆固醇簇的形成的反映。关键波动提供了一种在生理相关组成和温度下产生具有亚微米尺寸的脂质结构的机制。该项目的工作一直是Sarah Veatch博士，Sarah Keller博士和LMBB的NMR部分之间的合作研究。在这个项目的框架内，我们开发了用于检测不需要同位素标记的生物膜中有序脂质结构域的NMR工具。与NIH的Joshua Zimmerbergs实验室合作，这些工具已用于寻找完整流感病毒中有序的脂质域。在完整的病毒包膜和由提取的病毒脂质中制成的双层中，有序和无序脂质结构域共存的证据。正在研究病毒膜中有序脂质结构域形成的功能意义。 （ii）我们考虑了一个问题，即在脂质组成中是否可以通过双层曲率弹性应激的差异来完全阐明脂质组成的元视opsin I（mi）metarhopopsin II（MII）平衡的问题。添加了一系列具有单层曲率自发半径的六个脂质和弯曲弹性模量，以较高的浓度添加到基质脂质1-甲米酰基-2-烯酰基-SN-甘油-3-甘油-3-磷酸化磷酸（POPC）和MI-MIII Equilibrium和MI-MII Equilibrium和Mi-Mii Equilibrium的记录下，经记录了闪光照相，然后经过记录。每个脂质分子和膜疏水厚度的平均面积是从POPC中棕榈酸链的2H NMR阶参数曲线的测量得出的。对于具有不同水平头组甲基化水平的乙醇胺的系列，MI-MII平衡的变化与膜弹性性能的变化相关，如单层曲率自发半径，弯曲弹性模量和每个分子侧面面积所表达的。但是，在整个脂质系列中，弹性能量仅部分解释了这些变化。其他贡献与乙醇胺头组与蛋白质进行氢键的能力相关，与乙醇胺甲基化状态无关，并引入了多不饱和的SN-2烃链，并用油酸代替棕榈酸SN-1链。实验表明，在蛋白质周围的第一层脂质以及脂质蛋白质结构域中的膜弹性应激周围的脂质中，视紫红质与特定脂质物种相互作用的重要性。 我们探讨了GPCR视紫红素的表面是否应被视为均匀的，而周围的膜是连续的，或者是否应将特定的相互作用（尤其是与多不饱和脂质的特定相互作用）在宾夕法尼亚的激活中起作用。通过1H MAS NMR研究了视紫红素与多饱和脂质的缔合，并从视紫红质转移到脂质。结果表明，多紫红蛋白上的多种位点特异性相互作用。相关的脂质与矩阵的脂质快速交换，以毫秒或较短的时间尺度。所有视紫红质光蛋白光中间体都优先传递磁化强度到含DHA的脂质，表明相互作用更强。观察到元III视紫红质的最高率。与含DHA的脂质的相互作用是头组依赖性的，并且在磷脂酰胆碱，磷脂酰丝氨酸，磷脂酰乙醇胺的序列中的强度增加。 我们在完整的杆外段磁盘中使用1H MAS NMR在光激活时跟踪视紫红质水合的变化。数据表明，元视opsin-i与深色适应的视紫红质和其他光水解中间体有所不同。尽管这些实验不能直接确定蛋白质水合变化的位置，但是当相互作用既短距离（<5）和长寿命（1-10 ns）时，磁化转移最有效，这是通过蛋白质核心水合来满足的条件。激活后GPCR水合的变化使GPCR信号传导特别敏感地对影响水分子活性的环境因素敏感。同样的技术用于研究与Ninds Kenton Swartz的实验室合作的膜上膜的电压感应蛋白结构域的水合。 （iii）大麻素受体由于其在包括酒精中毒在内的健康和疾病中的作用而引起了很多关注。外周大麻素受体CB2参与免疫和激素反应。 CB2在大肠杆菌中表达为与麦芽糖结合蛋白和几个亲和力标签的融合。将融合裂解，并在洗涤剂存在下被Ni-NTA和链霉素亲和色谱纯化。比较了将CB2重构为脂质双层的几种方法（快速稀释，透析，去除吸收树脂的去污剂）。通过分析NMR，使用标记的脂质和CB2的荧光光谱，动态光散射以及蔗糖梯度离心研究了蛋白质脂质体的组成，大小和均匀性。蛋白质在纯化和重建过程中成功稳定，由洗涤剂，脂质以及配体的适当混合物。包含90％功能CB2的毫克的样品的制备成功。重构蛋白的结构完整性通过其对激动剂结合而激活G蛋白的能力得到了证实。我们开发了一种细菌发酵方案，用于生产稳定的同位素标记的大麻素受体CB2，用于通过核磁共振光谱法对该蛋白进行随后的结构研究。每升培养基的发酵过程产生了约2 mg的纯化，标记和功能性CB2。