NMR Investigations Of Cell Membrane Structure

我们在大肠杆菌中重组表达CB2与麦芽糖结合蛋白和几个亲和标签的融合。将CB2融合蛋白溶解、纯化、裂解，然后从裂解产物中再次纯化CB2。我们广泛研究了洗涤剂、脂类和大麻素配体对重组大麻素受体CB2稳定性的影响。这一努力产生了适用于广泛下游应用的全功能受体的制备和处理指南。我们证明了阴离子胆固醇衍生物，胆固醇半琥珀酸酯（CHS）和高亲和大麻素配体CP-55,940或SR-144,528的协同作用是有效稳定CB2在十二烷基麦芽糖苷(DDM)/ CHAPS洗涤剂溶液中的功能折叠所必需的。与CHS类似，带丝氨酸头基团（PS）的带负电荷磷脂在胶束中发挥了显著的稳定作用，而不带电荷的磷脂则不起作用。重组成脂质双分子层的纯化CB2保留了长达数周的功能，使该GPCR在生理相关条件下的高分辨率结构研究成为可能。

We express CB2 recombinantly in Escherichia coli as a fusion with maltose-binding protein and several affinity tags. The CB2-fusion protein is solubilized, purified, the fusion cleaved, and CB2 purified again from cleavage products. We extensively studied the effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2. The effort resulted in guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/ CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions. Reconstitution of functional CB2 at the level of milligrams, and concentration to a volume of 40 microliters, sufficient for structural studies by solid state NMR has been achieved. Functionality of the receptor was verified by ligand binding using radioactive ligands as well as deuterated ligands in combination with 2H-MAS NMR and by G protein activation studies using recombinantly produced G protein in a GTPgammaS radioactive assay. 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. Exploratory NMR experiments conducted on a 2-mg sample of homogeneously 13C- and 15N labeled CB2 and comparison of experimental results with simulated spectra obtained from the atomic coordinates of a CB2 model have demonstrated feasibility of the experimental concept. Specific isotopic labeling schemes by chemical labeling of amino acids as well by specific isotopic labeling of amino acids are under development to achieve the desired spectral resolution for structural analysis by NMR as well as EPR. The goal of these studies is to determine structural differences as a function of ligands that are bound to the receptor. Dimerization of GPCR has emerged as an essential mechanism regulating GPCR biosynthesis, maturation, ligand binding, coupling with G protein and downstream signaling in cell-signaling pathways. However, determining the oligomeric state of a GPCR in a membrane is challenging. It was explored if small angle neutron scattering (SANS) is a suitable tool to study the state of GPCR oligomerization at functional conditions of the receptor. Experiments were conducted with protonated bovine rhodopsin reconstituted into a perdeuterated lipid matrix. This yields maximal neutron scattering length density contrast between lipid and protein. Incoherent scattering of neutrons was minimized by conducting experiments in perdeuterated buffer. It was observed that the state of rhodopsin oligomerization in a lipid matrix at lipid-to-protein molar ratios near 500/1 depends on the state of photoactivation of the receptor. While dark-adapted rhodopsin was monomeric, bleached rhodopsin formed mostly dimers. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. We develop strategies for preparation of functional, recombinant CB2 and immobilization at solid interfaces. The successful deposition of CB2 was demonstrated by surface plasmon resonance. Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (MII), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether MII-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18:0-22:6n-3PE, 18:0-22:5n-6PE and the model lipid 18:1n-9-18:1n-9PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 Angstrom. Negative curvature elastic stress in membranes containing high concentrations of polyunsaturated PEs is very high. Release of even a small fraction of this stress from the layer of lipids surrounding the receptor is sufficient to shift the MI/MII equilibrium towards MII, the state that activates G protein. Furthermore, polyunsaturated bilayers have a hydrophobic thickness of about 27 A which has been determined to match the length of the hydrophobic transmembrane helices of rhodopsin. The data show that polyunsaturated lipids are important for class A GPCR activation, and we speculate that the rhodopsin model is particularly relevant for constitutive activity of GPCR and activation by weak agonists. We continue our studies on biophysical properties of the lipid matrix that are important for function of integral membrane proteins. In collaboration with laboratories that conduct molecular simulations, we explored the internal structure of the liquid ordered phase that forms in the presence of high cholesterol concentrations in membranes. The liquid ordered phase of a mixture of cholesterol and two lipids was shown to be itself inhomogeneous. Lateral segregation within the phase is observed, with regions of hexagonally packed saturated chains separated by interstitial regions enriched in cholesterol and unsaturated chains. The observed substructure explains existing experimental data and provides a focus for future efforts aimed at understanding the molecular scale structure of cell membranes. This picture of the phase provides an explanation for a number of experimental results, most of them obtained by NMR, which have until now lacked a consistent description in terms of a molecular model.