Docosahexaenoyl Chains Isomerize on the Sub-Nanosecond Time Scale

The dynamics of docosahexaenoyl acyl chains (DHA) in 18:0-22:6n3-PC bilayers were studied by 13C MAS NMR relaxation measurements. A Lipari−Szabo-type analysis yielded site-specific correlation times of DHA chain isomerization and C−H bond order parameters. It is concluded that DHA chains perform rapid isomerization with correlation times from 80 ps near the carbonyl group to 8 ps near the terminal methyl group. Spin−lattice relaxation rates remained unaltered after rhodopsin incorporation into the bilayers, indicating that the majority of lipids maintain their rapid chain isomerization in the presence of the protein. However, spin−spin relaxation rates revealed that rhodopsin increased motional correlation times of slow collective DHA motions

NOESY NMR Crosspeaks between Lipid Headgroups and Hydrocarbon Chains: Spin Diffusion or Molecular Disorder?

NOESY NMR Crosspeaks between Lipid Headgroups and Hydrocarbon Chains:  Spin Diffusion or Molecular Disorder

Lateral Lipid Diffusion Dominates NOESY Cross-Relaxation in Membranes

Lateral Lipid Diffusion Dominates NOESY Cross-Relaxation in Membrane

Polyunsaturated Fatty Acids in Lipid Bilayers: Intrinsic and Environmental Contributions to Their Unique Physical Properties

Polyunsaturated lipids are an essential component of biological membranes, influencing order and dynamics of lipids, protein−lipid interaction, and membrane transport properties. To gain an atomic level picture of the impact of polyunsaturation on membrane properties, quantum mechanical (QM) and empirical force field based calculations have been undertaken. The QM calculations of the torsional energy surface for rotation about vinyl−methylene bonds reveal low barriers to rotation, indicating an intrinsic propensity toward flexibility. Based on QM and experimental data, empirical force field parameters were developed for polyunsaturated lipids and applied in a 16 ns molecular dynamics (MD) simulation of a 1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC) lipid bilayer. The simulation results are in good agreement with experimental data, suggesting an unusually high degree of conformational flexibility of polyunsaturated hydrocarbon chains in membranes. The detailed analysis of chain conformation and dynamics by simulations is aiding the interpretation of experimental data and is useful for understanding the unique role of polyunsaturated lipids in biological membranes. The complete force field is included as Supporting Information and is available from http://www.pharmacy.umaryland.edu/faculty/amackere/research.html

Lipid−Rhodopsin Hydrophobic Mismatch Alters Rhodopsin Helical Content

The ability of photoactivated rhodopsin to achieve the enzymatically active metarhodopsin II conformation is exquisitely sensitive to bilayer hydrophobic thickness. The sensitivity of rhodopsin to the lipid matrix has been explained by the hydrophobic matching theory, which predicts that lipid bilayers adjust elastically to the hydrophobic length of transmembrane helices. Here, we examined if bilayer thickness adjusts to 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 mono-unsaturated phosphatidylcholines with 14−20 carbons per hydrocarbon chain. Changes of hydrocarbon chain length were measured by 2H NMR, and protein helical content was quantified by synchrotron radiation circular dichroism and conventional circular dichroism. Experiments were conducted on dark-adapted rhodopsin, the photo-intermediates 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. Helical content did not change measurably upon photoactivation. The increases of bilayer thickness and helicity of rhodopsin are accompanied by higher metarhodopsin II/metarhodopsin I ratios, faster rates of metarhodopsin II formation, an increase of tryptophan fluorescence, and higher temperatures of rhodopsin denaturation. The data suggest a surprising adaptability of this G protein-coupled membrane receptor to properties of the lipid matrix

Stabilizing effects of POPC:POPS (1∶1, w/w) in micelles.

<p>A, Lipids were added to DDM/CHAPS (0.1%/0.5%) micelles at concentrations indicated. Upon incubation samples were supplemented with POPC:POPS dissolved in 1% CHAPS so that the final protein-to-lipid ratio was the same in all samples (1∶500 mol/mol). Upon reconstitution on a mini-spin detergent-absorbent columns the activity of CB₂ was determined by the G protein activation assay and reported as % of the maximal activity measured for this series of samples. The liposomes-reconstituted receptor (0.1% CHS in micelles, POPC/POPS/CHS 60∶15:25 in liposomes) exhibiting the highest levels of activation in this experiment was used as an activity standard. B, Purified CB₂ in DDM/CHAPS/CHS micelles was captured on Ni-NTA, detergent buffer rapidly exchanged to DDM/CHAPS containing 0.4% of lipids of indicated composition, protein eluted with imidazole and liposome-reconstituted by rapid dilution. Functional activity of CB₂ reconstituted into POPC/POPS/CHS matrix is set as 100% of activity. Figures depict data ± SD (error bars) of duplicate determinations from representative experiments (n = 2-3).</p

Activation of G proteins by recombinant CB₂ in <i>E. coli</i> membranes expressing CB₂-130 and in CHO membranes expressing CB₂.

<p>4 ng of CB₂ was used in the assay and the figure depicts data ± S.D. (error bars) of duplicate determinations from representative experiments (n = 3).</p

Recovery of functional CB₂ in proteoliposomes prepared from various dominant detergents.

<p>Activity is presented as % of the maximal activity in the series (1% LDAO). The results shown represent data ± S.D. (error bars) of duplicate determinations from single representative experiments (out of three independently performed experiments, n = 3)</p

Stability of CB₂ in lipid bilayers.

<p>A, Temperature-induced unfolding of CB₂ in detergent micelles and lipid bilayers. For stability studies in micelles the purified CB₂-130 in TD buffer supplemented with 10 µM CP-55,940 was subjected to a temperature gradient from 4°C to 74°C at a rate of 1°C/min, 10 µg protein samples withdrawn at indicated time points, mixed with 100 µg lipids POPC/POPS (4∶1 w/w) in 1% CHAPS and diluted 110-fold into cold 10 mM MOPS buffer under vigorous stirring. The activity of CB₂ was analyzed by measuring the G protein activation rates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046290#s4" target="_blank">Materials and Methods</a>. For measurement of thermostability in lipid bilayers either CB₂-proteoliposomes or membrane preparations harboring fusion CB₂-130 were suspended in 10 mM MOPS buffer at a concentration of CB₂ 0.5 ng/µL, subjected to treatment with linear temperature gradient, and analyzed by G protein activation assay. Dotted line depicts the temperature gradient profile. Figure depicts data ± S.D (error bars) of duplicate measurements from representative experiments (n = 3). B, Temperature stability of CB₂ in proteoliposomes and <i>E. coli</i> membranes. Either purified CB₂ receptor reconstituted into POPC:POPS:CHS bilayers or fusion CB₂-130 in <i>E. coli</i> membranes was incubated for 30 min at the temperatures indicated, and the G protein activation assay performed. 4 ng of CB₂ was used in every reaction and measurements were performed upon addition of 2 µM of CP-55,940 to all samples. Data ± S.D. (error bars) of duplicate measurements from representative experiments (n = 3) are presented.</p

Summary of experimental strategy.

<p>A, Testing stabilizing effects of detergents, ligands and lipids. •Efficient solubilization of the fusion CB₂ protein from <i>E. coli</i> membranes. Over 40 different detergents and mixtures of detergents were compared for their efficiency in solubilizing fusion CB₂ from membranes, and the detergent mixture that performed best was selected for a routine receptor purification protocol. •Optimization of the liposome-reconstitution procedure. A screening of lipid-solubilizing detergents was performed with a goal of maximizing the yield of functionally reconstituted receptor. •Application of the G protein activation test to the analysis of the structural stability of CB₂ in micelles following its reconstitution into liposomes. •Screening for stabilizers for CB₂ and characterization of stability of the purified receptor. (i)Stabilization of CB₂ in micelles by CHS, ligands and phospholipids. (ii)Ligand binding studies by solid-state NMR. (iii)Characterization of stability of CB₂ in lipid bilayers. B, Comparison of stabilizing effects of two lipids at two different temperatures.</p