(i) 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. 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 critical behavior. 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 that modulate protein function. This has been a collaborative research effort between Dr. Sarah Veatch, Dr. Sarah Keller, and the NMR Section of LMBB. In its framework 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 techniques were used to search for ordered lipid domains in intact influenza virus and of lipid extracts thereof. It was observed that ordered and disordered lipid domains coexist in the physiological range of temperatures for both the intact virus envelope and the lipid extracts. At ambient temperature, a significant portion of lipids is in a gel phase whose fraction reversibly increased with cholesterol depletion. The existence of gel phase domains at room temperature was confirmed by lipid diffusion measurements using 1H MAS NMR with application of pulsed magnetic field gradients. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. At temperatures of 37oC and higher the fraction of ordered lipids is small and independent of the presence of native proteins. Formation of ordered lipid phases is likely to be important for viral stability. ? ? (ii) The hydrophobic matching theory predicts that lipid bilayers adjust elastically to the hydrophobic length of transmembrane helices. This theory has been used to explain the effects of membrane composition on protein function for a number of transmembrane proteins, including the dim-light receptor rhodopsin. We examined the basic tenet of this theory by determining if bilayer thickness truly changes to match the length of the protein or if the protein alters its conformation to adapt to the bilayer. Purified bovine rhodopsin was reconstituted into a series of monounsaturated phosphatidylcholines with 14 to 20 carbons per hydrocarbon chain. Changes of hydrocarbon chain length were measured by 2H-NMR and protein helical content quantified by synchrotron radiation circular dichroism. Experiments were conducted on dark-adapted rhodopsin, the photointermediates metarhodopsin I/II/III, and opsin. Changes of bilayer thickness upon rhodopsin incorporation and photoactivation were mostly absent. In contrast, the helical content of rhodopsin increased with membrane hydrophobic thickness, equivalent to elongation of transmembrane helices by 1-2 turns. Helical content was the same for all photointermediates. The data indicate that transmembrane helices of rhodopsin have an underappreciated plasticity for matching bilayer thickness. The bilayer-induced structural changes persist after photoactivation. Altered helical content of rhodopsin appears to be directly responsible for changes in the level of activation and for activation rates. ? 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. It was shown that poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface and that interaction strength is mudulated by lipid headgroups and by photoactivation of rhodopsin. Rhodopsin-associated lipids are in fast exchange with lipids in the bulk of the matrix on a timescale of milliseconds or shorter.? 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 and long-lived (1-10 ns), conditions best satisfied by hydration of the protein core. Molecular simulations with photoactivation of the receptor conducted by our collaborators at IBM, supported by the Blue Gene project, had suggested such a water influx into the core of the protein upon metarhodopsin-I formation. The internal waters interact with several amino acid residues that are highly conserved in the GPCR family, indicating that changes in internal hydration may be important during the activation of other GPCRs as well. Changes of GPCR hydration upon activation make GPCR signaling particularly sensitive to environmental factors. ? ? (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. While structural characterization of cannabinoid receptors including studies of their interaction with ligands is highly desirable, such efforts are currently hampered by the lack of sufficient quantities of purified, functional protein. The goal of this project is to develop methods for production and functional reconstitution of CB2 at the milligram level for investigation of lipid-protein interaction and protein structural studies by NMR. 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 Strep-tactin affinity chromatography in the presence of detergents. Several procedures for reconstitution of CB2 into lipid bilayers (rapid dilution, dialysis, removal of detergents with absorbing resins) were compared. The ability of reconstituted CB2-containing proteoliposomes to activate G-proteins in response to agonist binding was studied as a function of detergents, lipids, and of CB2 concentration. 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 as well as ligand. Preparation of samples that contain up to one milligram of mostly functional CB2 was successful. Currently fermentation conditions are optimized for expression of labeled protein.
Showing the most recent 10 out of 45 publications
