Klaus Gawrisch, PhD, Section Chief
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health
5625 Fishers Lane, Room 3N07:MSC 9410
Bethesda MD 20892-9410
telephone: +1 301 594 3750
fax: +1 301 594 0035
e-mail: klausg@mail.nih.gov
We conduct structural and functional studies on reconstituted G-protein coupled membrane receptors (GPCR). The goal is to investigate GPCR at functional conditions in a fluid lipid matrix with a biologically relevant composition of lipids. In particular neuronal GPCR are located in membranes containing high concentrations of poly-unsaturated fatty acids (PUFA). The mechanisms by which polyunsaturated lipid species may influence biological function at the molecular level have attracted considerable attention. Two mechanisms of PUFA action on proteins are considered: on one hand, PUFA may influence function by altering biophysical properties of the lipid matrix. Such action most likely occurs in neuronal membranes with high PUFA content like synaptosomal membranes and the retina. On the other hand, PUFA and their derivatives may act as ligands that interact with membrane pro¬teins specifically.
Bacterial expression, purification, and functional reconstitution of the peripheral cannabi-noid receptor, CB2
Yeliseev, Krepkiy, Vukoti, Kimura, Zoubak, Teague
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 pro-tein 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 methods for reconsti-tution of CB2 into lipid bilayers (rapid dilution, dialysis, removal of detergents with absorbing resins) were compared. Composition, size, and homogeneity of proteoliposomes were investi¬gated 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. Prepa-ration of samples that contain milligrams of mostly functional CB2 was successful. The struc¬tural integrity of the reconstituted protein was confirmed by its ability to activate G-protein in response to agonist binding.
Yeliseev A, Zoubak L, Gawrisch K. Use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor.
Prot Expr Purific 53: 153-63, 2007.
Krepkiy D, Gawrisch K, Yeliseev A. Expression and purification of CB2 for NMR studies in mi-cellar solution.
Protein Peptide Lett 14: 1031-7, 2007.
Key prior publications:
Yeliseev A, Wong K, Soubias O, Gawrisch K. Expression of human peripheral cannabinoid re-ceptor for structural studies.
Protein Sci 14, 2638-53, 2005.
Krepkiy D, Wong K, Gawrisch K, Yeliseev A. Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2.
Prot Expr Purific 49: 60-70, 2006.
Rhodopsin interaction with the lipid matrix
Soubias, Teague, Hines, Niu, Mitchell, Grossfield, Pitman
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 heli-cal 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 ther-mal denaturation, and of intrinsic tryptophan fluorescence. This is contrary to the current para¬digm that hydrophobic matching involves primarily an adjustment of the lipid matrix to the pro¬tein. 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 polyunsatu-rated 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 rhodop-sin to lipid. It was shown that poly- and monounsaturated lipids interact specifically with differ¬ent 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 ob-served for Meta-III rhodopsin. Interactions with DHA-containing lipids are headgroup dependent and strength increased in the sequence phosphatidylcholine, phosphatidylserine, phosphati-dylethanolamine.
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, magnetiza-tion transfer is most efficient when the interactions are both short-range (< 5 Å) 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.
Publications 2007-2008:
Soubias O, Niu S-L, Mitchell DC, Gawrisch K. Lipid-rhodopsin hydrophobic mismatch alters rhodopsin helical content.
J Am Chem Soc 130: 12465-71, 2008.
Grossfield A, Pitman MC, Feller SL, Soubias O, Gawrisch K. Internal hydration increases during activation of the G protein-coupled receptor rhodopsin.
J Membr Biol 381: 478-86, 2008.
Key prior publications:
Soubias O, Teague WE, Gawrisch K. Evidence for specificity in lipid-rhodopsin interactions.
J Biol Chem 281: 33233-24, 2006.
Soubias O, Gawrisch K. Probing specific lipid-protein interaction by saturation transfer difference NMR spectroscopy.
J Am Chem Soc 127: 13 110-1, 2005.
Properties of polyunsaturated bilayers and their influence on membrane protein function
Soubias, Mihailescu, Teague, Feller
The dynamics of docosahexaenoyl acyl chains (DHA) in 1 8:0-22:6n3-PC bilayers near rhodopsin were studied by 13C MAS NMR relaxation measurements. It is concluded that DHA chains perform rapid isomerization with correlation times on the timescale of 100 picoseconds or less. The DHA chain explores its entire conformational space within 10 nanoseconds. The great ma¬jority of lipids maintain their rapid chain isomerization in the presence of rhodopsin. However, spin-spin relaxation rates revealed that rhodopsin increased motional correlation times and/or amplitudes of slow collective DHA motions. By NMR and diffraction studies we obtained evi¬dence that the low molecular order in bilayers with high DHA content is a direct consequence of high conformational flexibility and of rapid structural conversions of DHA chains. This flexibil¬ity is caused by extremely low potential barriers for changes of dihedral bond angles in vinyl bonds. Low potential barriers permit the polyunsaturated chains to rapidly change conformation without significant energetic penalty. Flexibility and adaptability of polyunsaturated fatty acids impart unique elastic properties on lipid bilayers which are likely to ease conformational transi¬tions of GPCR upon their activation as it was observed experimentally. The distribution of satu¬rated stearic acid (18:0) and polyunsaturated DHA in the mixed chain phosphatidylcholine 18:0- 22:6n3-PC was measured by neutro scattering. It was observed that DHA segments are located with higher probability near the lipid/water interface, while the segments of saturated chains lo¬cate with higher probability in the bilayer center. DHA chains in phosphatidylethanolamines fa¬cilitate formation of inverted hexagonal phases, an indication for curvature elastic stress in the lipid matrix which is known to shift the structural equilibrium between the photointermediates of rhodopsin.
Gawrisch K, Soubias O. Structure and dynamics of polyunsaturated hydrocarbon chains in lipid bilayers – significance for GPCR function.
Chem Phys Lipids 153: 64-75, 2008.
Soubias O, Gawrisch K. Docosahexaenoyl chains isomerize on the sub-nanosecond time scale.
J Am Chem Soc 129: 6678-9, 2007.
Key prior publications:
Mihailescu M, Gawrisch K. The structure of polyunsaturated lipid bilayers important for rhodopsin function: a neutron diffraction study.
Biophys J 90: L04-6, 2006.
Feller SE, Gawrisch K. Properties of docosahexaenoic-acid-containing lipids and their influence on the function of rhodopsin.
Curr Opin Struct Biol 15, 416-22, 2005.
Koenig BW, Gawrisch K. Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase.
Biochim Biophys Acta 1715: 65-70, 2005.
Pitman MC, Suits F, Gawrisch K, Feller SE. Molecular dynamics investigation of dynamical properties of phosphatidylethanolamine lipid bilayers.
J Chem Phys 122: 244715, 2005.
Raft formation in biomembranes
Veatch, Polozov, Scheidt, Soubias, Keller, Zimmerberg
We obtained evidence for critical behavior in cholesterol-rich model membranes that form coex-isting liquid ordered and disordered phases which have been linked to raft formation in biologi¬cal membranes. Deuterium NMR was used to evaluate phase boundaries in cholesterol contain¬ing ternary lipid membranes. The precise thermodynamic description of phase behavior permit¬ted 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 pro¬ject we developed NMR tools for detection of ordered lipid domains in biological membranes that do not require isotopic labeling. In collaboration with Dr. Joshua Zimmerberg’s 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 enve¬lope 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.
Polozov IV, Bezrukov L, Gawrisch K, Zimmerberg J. Progressive ordering with decreasing temperature of the phospholipids of influenza virus.
Nat Chem Biol 4:248-55, 2008.
Veatch SL, Soubias O, Keller SL, Gawrisch K. Critical fluctuations in domain-forming lipid mixtures.
Proc Natl Acad Sci USA 104: 17650-5, 2007.
Polozov IV, Gawrisch K. NMR detection of lipid domains.
Methods Mol Biol 398: 107-26, 2007.
Key prior publications:
Polozov IV, Gawrisch K. Characterization of the liquid-ordered state by proton MAS NMR.
Biophys J 90: 2051-61, 2006.
Veatch SL, Gawrisch K, Keller SL. Closed-loop miscibility gap and quantitative tie-lines in ternary membranes containing diphytanoyl PC.
Biophys J 90: 4428-36, 2006.
Scheidt HA, Huster D, Gawrisch K. Diffusion of cholesterol and its precursors in lipid mem-branes studied by 1H pulsed field gradient magic angle spinning NMR.
Biophys J 89: 2504-12, 2005.
GPCR reconstitution into bilayers supported by nanoporous substrates
Soubias, Gaede, Yeliseev, Teague
We developed reagents and methods for formation of tubular, single lipid bilayers containing high concentrations of GPCR that cover the pore walls of porous anodic aluminum oxide (AAO) filters. One square centimeter of the filter material with a thickness of 60 micrometers yields up to 500 cm2 of oriented membranes, which is sufficient for multinuclear solid state NMR studies. By solid state NMR methods we determined that the membranes are separated from the support by a closed and stable aqueous cushion. The inner surface of the lipid tubules is freely accessible from an outside solution. The aluminum oxide-based support provides the advantage of high flow rates to exchange solutions, efficient particle retention, rigid, uniform surface, and transpar¬ency (when wet). The GPCRs, rhodopsin, purified from natural sources, as well as the recombi¬nant peripheral cannabinoid receptor, CB2, expressed in E. coli were incorporated into the tubu¬lar bilayers in functional form. Reconstitution of bovine rhodopsin into AAO filters did not inter¬fere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-Vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTP-y-S bind¬ing assay. Lipid-rhodopsin interaction was investigated by 2H NMR on sn-1-, or sn-2 chain per¬deuterated 1 -stearoyl-2-docosahexaenoyl-sn-glycero-3 -phospholine as matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed spectral density of motions with correlation times in the range from nano- to microseconds, detected as significant reduction of spin-spin relaxation times. The change of lipid chain order parameters due to interaction with rhodopsin was insignificant.
Publication 2008:
Soubias O, Niu S-L, Mitchell DC, Gawrisch K. Lipid-rhodopsin hydrophobic mismatch alters rhodopsin helical content
J Am Chem Soc 130: 12465-71, 2008.
Key prior publications:
Soubias O, Polozov IV, Teague WE, Yeliseev AA, Gawrisch K. Functional reconstitution of rhodopsin into tubular lipid bilayers supported by nanoporous media.
Biochemistry 45: 15583-90, 2006.
Yeliseev A, Wong K, Soubias O, Gawrisch K. Expression of human peripheral cannabinoid re-ceptor for structural studies.
Protein Sci 14: 2638-53, 2005.
Gaede HC, Luckett KM, Polozov IV, Gawrisch K. Multinuclear NMR studies of single lipid bi-layers supported in cylindrical aluminum oxide nanopores.
Langmuir 20: 7711-9, 2004.
Updated: May 2009