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了膜组成对乙醇分配到脂质双分子层的影响。研究了一系列不同组成的模型膜。膜暴露在生理浓度为20毫米的乙醇中。膜的浓度为20 wt%，大致与组织中的值相对应。分配取决于脂水界面上极性基团的化学性质。与磷脂酰胆碱相比，含有磷脂酰甘油、磷脂酰丝氨酸和鞘磷脂的脂质分配系数增强，而含有磷脂酰乙醇胺的脂质分配系数降低。摩尔分配系数与膜的疏水体积无关。这一观察结果与我们之前发表的核磁共振结果一致，核磁共振结果表明乙醇几乎完全存在于膜-水界面中。当乙醇在水中浓度为20 mM时，脂/水界面处的乙醇浓度在30 - 15 mM范围内，对应于每100-250个脂质中有一个乙醇分子。