Walter E. Teague

Evidence for Specificity in Lipid-Rhodopsin Interactions

The interaction of bovine 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 lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg2+], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K′ was defined asEquation 1 K ′ = [ ATP ] [ Arg ] [ ADP ] [ PArg ] where each reactant represents the sum of all the ionic and metal complex species. The K′ at pH 7.0, 1.0 mm free [Mg2+], and 0.25 m ionic strength was 29.91 ± 0.59, 33.44 ± 0.46, 35.44 ± 0.71, 39.64 ± 0.74, and 45.19 ± 0.65 (n = 8) at 40, 33, 25, 15, and 5 °C, respectively. The standard apparent enthalpy (ΔH°′) is −8.19 kJ mol−1, and the corresponding standard apparent entropy of the reaction (ΔS°′) is + 2.2 J K−1mol−1 in the direction of ATP formation at pH 7.0, free [Mg2+] =1.0 mm, ionic strength (I) =0.25 mat 25 °C. We further show that the magnitude of transformed Gibbs energy (ΔG°′) of −8.89 kJ mol−1 is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TΔS°′ term (+0.66 kJ mol−1). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.

Controlling GPCR Rhodopsin Function by Small, Physiologically Relevant Changes in Bilayer Hydrophobic Thickness

It was established previously that the G protein-coupled membrane receptor rhodopsin has transmembrane helices which match a hydrophobic bilayer thickness of 27±1 A. Here we demonstrate that small changes of bilayer thickness of ±2 A about that match point translate in the considerable changes of rhodopsin activation measured as the metarhodopsin I (MI)/metarhodopsin II (MII) equilibrium. We observed a biphasic behavior of the MI/MII equilibrium, with a sharp decline towards MI from 25-27 A followed by a rapid increase of MII from 27-29 A. Results are qualitatively identical for thickness changes induced by mixing of 16:0-16:1 PC and 18:0-18:1 PC, or 16:1-16:1 PC and 18:1-18:1 PC, or addition of 0-30 mol% cholesterol to 16:0-16:1 PC. The biphasic behavior was observed regardless of lipids used to alter bilayer hydrophobic thickness suggesting a relationship between small changes in hydrophobic thickness and rhodopsin function. It strongly favors an explanation based on a change of elastic stresses in lipid bilayers upon the transition from negative curvature in lipid monolayers near the protein below 27 A hydrophobic thickness to positive monolayer curvature above the match point. A continuum elastic model of the membrane, including the effect of lipid monolayer curvature near the protein, predicts membrane mediated clustering of rhodopsin and stabilization of the MI photointermediate at the matching point. Small, physiologically relevant changes in cholesterol content of bilayers with a thickness in the physiologically relevant range do drastically down- or up regulate the amount of MII which is the state that activates G protein.

Cholesterol Enhances or Reduces Metarhodopsin II Formation Depending on Bilayer Thickness

Cholesterol is one of the most efficient modulators of G Protein-Coupled Receptor (GPCR) function. We monitored the effect of cholesterol on rhodopsin function for a set of bilayers with different hydrophobic thickness. Surprisingly, cholesterol shifts the Metarhodopsin-I (MI)/Metarhodopsin-II (MII) equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (2.7 nm), and to MI for thicker bilayers. In previous work conducted on rod outer segment disks and model membranes, increasing cholesterol concentration always shifted the equilibrium towards MI. It was proposed that the cholesterol effect is primarily related to a tighter packing of lipid hydrocarbon chains which generates a less permissive environment for the formation of MII. To gain deeper insights into mechanisms, we followed changes in lipid-rhodopsin interaction by 2H NMR using deuterated lipids. It was reported by us and the Brown laboratory that an increase of bilayer hydrophobic thickness in the absence of cholesterol favors MII with a turnover to MI for bilayers that are very thick. Indeed, the cholesterol-induced shifts towards MII for thinner membranes correlated nicely with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR suggesting that the increase in bilayer thickness by cholesterol plays a major role in controlling the energetics of the MI-MII equilibrium. Furthermore, changes in average lipid order parameters due to the presence of rhodopsin were much larger in cholesterol-containing membranes than in cholesterol-free membranes suggesting strongly that the perturbation in the lipid matrix from protein insertion reaches much further away from the protein. This is expected for membranes that are stiffer due to the presence of cholesterol. The consequences of these findings for lipid mediated shifts in rhodopsin function and rhodopsin-rhodopsin interactions will be discussed.

Lipid Lateral Diffusion and Hydrocarbon Chain Order

Recent advances in molecular simulations have stimulated new discussions on the molecular mechanisms of lateral diffusion of lipids in fluid bilayers. To enable a deeper comparison between theory and experiments, we conducted a systematic study of lateral diffusion rates for a series of phosphatidylcholines with 14, 16, 18 and 20 hydrocarbons per chain over the temperature range from 10 to 600C. Bilayers of the pure lipids as well as binary mixtures with 30 mol% cholesterol were investigated. Diffusion coefficients were measured by 1H MAS NMR with application of pulsed magnetic field gradients. All lipids had saturated, perdeuterated sn-1 chains that enabled measurement of chain order parameters by 2H NMR. From the average chain order parameters effective hydrophobic thicknesses of bilayers and lateral areas per lipid molecule were calculated. This enabled a quantitative comparison of experimentally determined and calculated lateral diffusion rates as a function of chain length, cholesterol content and temperature using a free-volume model of lateral diffusion. The implications of this comparison for models of lateral lipid diffusion will be discussed.