NMR Investigations Of Cell Membrane Structure

细胞膜结构的核磁共振研究

• 批准号: 7963820
• 负责人: KLAUS GAWRISCH
• 金额: $ 219.05万
• 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/7963820
• 关键词: Affinity; Affinity Chromatography; Agonist; Agreement; Alcoholism; Attention; Behavior; Binding; Binding Sites; Biological; Biological Process; CNR2 gene; Cannabinoids; Cell Membrane Structures; Centrifugation; Chemicals; Cholesterol; Cleaved cell; Collaborations; Core Protein; Coupled; Data; Detection; Detergents; Deuterium; Dialysis procedure; Diffusion; Dimensions; Disease; Docosahexaenoic Acids; Environmental Risk Factor; Equilibrium; Escherichia coli; Ethanol; Excision; Fluorescence; Fluorescence Spectroscopy; GTP-Binding Proteins; Gas Chromatography; Glycerol; Health; Hormonal; Hydration status; Immune; Investigation; Label; Laboratories; Lateral; Lecithin; Life; Ligands; Link; Lipid Bilayers; Lipids; Liquid substance; Location; Measures; Membrane; Membrane Lipids; Methods; Molecular; National Institute of Neurological Disorders and Stroke; Nature; Neurons; Partition Coefficient; Peripheral; Phase; Phosphatidyl glycerol; Phosphatidylethanolamine; Phosphatidylglycerols; Phosphatidylserines; Physiologic pulse; Physiological; Plant Resins; Play; Polyunsaturated Fatty Acids; Preparation; Property; Proteins; Publishing; Receptor Signaling; Relaxation; Research; Rhodopsin; Role; Sampling; Series; Site; Sphingomyelins; Structure; Sucrose; Surface; System; Tail; Techniques; Temperature; Tertiary Protein Structure; Thermodynamics; Thick; Time; Tissues; Tryptophan; United States National Institutes of Health; Viral; Water; Work; cannabinoid receptor; flexibility; influenzavirus; interest; light scattering; lipid disorder; magnetic field; maltose-binding protein; membrane model; metarhodopsin I; metarhodopsin II; milligram; millisecond; monounsaturated fat; phosphatidylethanolamine; photoactivation; polyunsaturated fat; proteoliposomes; receptor; reconstitution; research study; response; rod outer segment disc; solid state nuclear magnetic resonance; submicron; tool; virus envelope; voltage

(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) Our experiments have shown that rhodopsin adjusts its helical content to bilayer thickness while there is little if any adjustment in the thickness of the lipid matrix to the protein. Changes of helical content are continuous with bilayer thickness. They are paralleled by shifts in the balance of rhodopsin photointermediates, the rates of Metarhodopsin-II formation, the temperature of thermal denaturation, and of intrinsic tryptophan fluorescence. This is contrary to the current paradigm that hydrophobic matching involves primarily an adjustment of the lipid matrix to the protein. The observation is likely to be applicable to the entire class of G-protein-coupled membrane receptors. 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 mostly 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. The widely-used hydrophobic cannabinoid ligand CP-55,940 partitions with high efficiency into biomembranes. We studied location, orientation, and dynamics of CP-55,940 in POPC bilayers by solid-state NMR. Chemical-shift perturbation of POPC from the aromatic ring-current effect, as well as 1H NMR cross-relaxation rates, locate the hydroxyphenyl ring of the ligand near the lipid glycerol, carbonyls, and upper acyl-chain methylenes. Order parameters of the hydroxyphenyl ring determined by the 1H-13C DIPSHIFT experiment indicate that the bond between the hydroxyphenyl and the hydroxycyclohexyl rings is oriented perpendicular to the bilayer normal. 2H NMR order parameters of the nonyl tail are very low indicating that the hydrophobic chain maintains a high level of conformational flexibility in the membrane. Lateral diffusion rates of CP-55,940 and POPC were measured by 1H MAS NMR with pulsed magnetic field gradients. The rate of CP-55,940 diffusion is comparable to the rate of lipid diffusion. The magnitude of cross-relaxation and diffusion rates suggests that associations between CP-55,940 and lipids are with lifetimes of a fraction of a microsecond. CP-55,940 with its flexible hydrophobic tail may efficiently approach the binding site of the cannabinoid receptor from the lipid-water interface by lateral diffusion.

（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）我们的实验表明，视紫红质将其螺旋含量调节到双层厚度，而脂质基质的厚度几乎没有任何调整到蛋白质的厚度。螺旋含量的变化与双层厚度连续。它们与视紫红蛋白光中间体的平衡，元视opsin-II形成的速率，热变性温度和内在色氨酸荧光的速度相似。这与当前的范式相反，疏水性匹配主要涉及对蛋白质脂质基质的调整。该观察结果可能适用于整个G蛋白偶联的膜受体。 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。准备含有毫克的样品的准备大多数功能性CB2是成功的。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。 广泛使用的疏水性大麻素配体CP-55,940分区对生物膜效率高。我们研究了通过固态NMR在POPC双层中研究CP-55,940的位置，方向和动力学。 POPC从芳族环电流效应以及1H NMR交叉释放速率中的化学班次扰动，在脂质甘油，羰基和上酰基链甲基甲基甲基的脂质甘油，羰基和上甲基甲基甲基甲基苯甲酸，甲基苯甲酸，甲基苯甲酸甲基苯基附近定位羟基苯基环。通过1H-13C二速实验确定的羟基苯基环的顺序参数表明，羟基苯基与羟基环己基环之间的键是垂直于双层的正常情况。 Nonyl尾巴的2H NMR顺序参数非常低，表明疏水链在膜上保持高构象的柔韧性。用脉冲磁场梯度通过1H MAS NMR测量CP-55,940和POPC的横向扩散速率。 CP-55,940扩散的速率与脂质扩散速率相当。交叉解释和扩散速率的大小表明，CP-55,940和脂质之间的关联与微秒的寿命相关。 CP-55,940及其柔性疏水尾部可以通过侧向扩散从脂质 - 水界面有效接近大麻素受体的结合位点。