NMR Investigations Of Cell Membrane Structure

我们获得了富含胆固醇的模型膜的关键行为的证据，这些模型膜形成共存的有序和无序的液体相，这与生物膜中的筏形形成有关。采用核磁共振氘谱法评价含胆固醇三元脂质膜的相界。相行为的精确热力学描述允许预测临界行为发生时的成分和温度。核磁共振共振在临界点附近显著扩大，证实了它们的存在。展宽归因于在微秒时间尺度上链序调制引起的自旋-自旋弛豫率的增加。我们推测光谱展宽是脂质-胆固醇簇形成的反应，其寿命为微秒级。临界波动提供了在生理相关成分和温度下产生亚微米尺寸脂质结构的机制。这个项目的工作是由Sarah Veatch博士、Sarah Keller博士和LMBB的核磁共振部门合作研究的成果。在这个项目的框架中，我们开发了NMR工具，用于检测生物膜中不需要同位素标记的有序脂质结构域。在与美国国立卫生研究院Joshua zimmerberg博士实验室的合作中，这些工具已被用于在完整的流感病毒中寻找有序的脂质结构域。在完整的病毒包膜和由提取的病毒脂质制成的双层中，获得了有序和无序脂质结构域共存的证据。病毒膜中有序脂质结构域形成的功能意义正在研究中。

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. Broadening 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. By 1H-MAS-NOESY spectroscopy with coherence selection by application of pulsed magnetic field gradients we investigated interaction of water with POPC bilayers. Interpretation of results was aided by neutron scattering experiments conducted at identical experimental conditions to obtain the water density distribution function over bilayers. Analysis showed that water molecules interact almost exclusively with sites of the lipid-water interface, including choline-, phosphate-, glycerol-, and carbonyl groups. Lifetime of lipid-water associations is rather short, on the order of 100 ps. The weak crosspeaks between water and hydrophobic methylene protons could be as much the result of infrequent chain upturns towards the lipid-water interface as the result of an occasional deep penetration of water molecules into the hydrophobic core. The negligible water content in the bilayer center suggests that water penetration through the hydrophobic core of bilayers must be very rapid. (ii) It was 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. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing bovine rhodopsin and lipids with polyunsaturated, docosahexaenoyl (DHA) and monounsaturated oleoyl chains. Rhodopsin was photoactivated in the spinning MAS rotor and magnetization transfer followed as a function of time after photoactivation. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for meta-III rhodopsin. Rates of magnetization transfer from protein to DHA are lipid headgroup dependent and increased in the sequence PC, PS, PE. In experiments conducted as a function of DHA content, it was shown that poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rhodopsin-associated lipids are in fast exchange with lipids in the bulk of the matrix on a timescale of milliseconds or shorter. The dynamics of docosahexaenoyl acyl chains (DHA) in 18:0-22:6n3-PC bilayers near rhodopsin were studied by 13C MAS NMR relaxation measurements. It is concluded that DHA chains perform rapid isomerization with correlation times from 80 ps near the carbonyl group to 8 ps near the terminal methyl group. The DHA chain explores its entire conformational space within 10 nanoseconds. Spin-lattice relaxation rates remained unaltered after rhodopsin incorporation into the bilayers, indicating that the great majority of lipids maintain their rapid chain isomerization in the presence of the protein. However, spin-spin relaxation rates revealed that rhodopsin increased motional correlation times and/or amplitudes of slow collective DHA motions. Experiments on reconstituted rhodopsin benefited greatly from the use of porous aluminum oxide filters for preparation of solid supported, single lipid bilayers at densities sufficient for NMR structural studies. The single, tubular membranes line the inner surface of pores in anodic aluminium oxide (AAO), permitting easy exchange of buffers to control pH and ionic strength, but also delivery of ligands. The novel method yields oriented, detergent-free bilayers that are essentially unperturbed by the solid support. Rhodopsin incorporation increased mosaic spread of bilayer orientation, but changes of lipid hydrocarbon chain order were negligible. Photoactivation of rhodopsin as well as G-protein binding in AAO pores were indistinguishable from rhodopsin function in unsupported unilamellar liposomes. (iii) We previously reported feasibility of bacterial expression of functional human peripheral cannabinoid receptor CB2 as fusion with the maltose binding protein, thioredoxin A, and a decahistidine tag. In order to remove the expression tags and to facilitate purification of CB2, other fusion constructs containing a second (internal) decahistidine tag and/or a Strep-tag were generated. The CB2-fusion inserted into the cytoplasmic membrane of E. coli upon expression. Expression levels of the recombinant receptor in E. coli BL21(DE3) cells were as high as 1-2 mg per liter of bacterial culture. The recombinant receptor was ligand binding-competent, and activated cognate G-proteins in an in vitro coupled assay. The fusion CB2-125 protein was purified by immobilized metal affinity chromatography on a Ni-NTA resin. The fusion was cleaved with a Tobacco Etch Virus (Tev) protease and purified to yield milligram quantities of highly enriched CB2. Purification to over 90% homogeneity of the resulting CB2, containing an N-terminal Strep-tag was achieved by affinity chromatography on a StrepTactin resin. Circular dichroism spectroscopy on purified CB2 in micelles indicated high -helical content. The expression and purification protocol allows for production of large (milligram) quantities of functional peripheral cannabinoid receptor, suitable for subsequent structural characterization. Preliminary results on reconstitution of CB2 into lipid bilayers indicate that a significant fraction of reconstituted CB2 retained its ligand-binding properties and is G-protein activation competent.