NMR Investigations Of Cell Membrane Structure

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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 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. 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. To reduce the size of samples with precious isotopically labeled GPCR, we collaborate on application of dynamic nuclear polarization (DNP) techniques to our samples that raise sensitivity by one to two orders of magnitude. 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. In collaboration with the Laboratory of Martin Caffrey, Trinity College, Dublin, Ireland, we studying the structure of lipoprotein signal peptidase II (LspA), a membrane protein which is a key enzyme in Pseudomonas aeruginosa. Its function is to release signal peptides from prolipoproteins. The goal of the project is to gather structural information related to LspA inhibition and lipoprotein processing. Both solution-state NMR experiments on LspA in detergent micelles and solid-state NMR experiments on LspA reconstituted into a monoolein cubic phase are conducted. Spectra of both the apo form of LspA as well as the globomycin-bound form were obtained. 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.