Daniel Huster

Water permeability of polyunsaturated lipid membranes measured by 17O NMR.

Diffusion-controlled water permeation across bilayers of polyunsaturated phospholipids was measured by 17O nuclear magnetic resonance. In 100-nm extruded liposomes containing 50 mM MnCl2, water exchange between internal and external solutions was monitored via changes in the linewidth of the 17O water resonance of external water. Liposome size and shape were characterized by light scattering methods and determination of liposome trapped volume. At 25 degrees C, the following water permeability coefficients were determined: 18:0-18:1n-9 PC, 155 +/- 24 microns/s; 18:0-18:3n-3 PC, 330 +/- 88 microns/s; and 18:0-22:6n-3 PC, 412 +/- 91 microns/s. The addition of 1 M ethanol reduced permeability coefficients to 66 +/- 15 microns/s for 18:0-18:1n-9 PC and to 239 +/- 67 microns/s for 18:0-22:6n-3 PC. Furthermore, the addition of 50 mol% 18:1n-9-18:1n-9 PE reduced the water permeability from 122 +/- 21 microns/s for pure 18:1n-9-18:1n-9 PC to 74 +/- 15 microns/s for the mixture. The significant increase in water permeation for membranes with polyunsaturated hydrocarbon chains correlates with looser packing of polyunsaturated lipids at the lipid-water interface and the suggested deeper penetration of water into these bilayers. Ethanol may block water diffusion pathways by occupying points of water entry into bilayers at the interface. The addition of dioleoylphosphatidylethanolamine increases lipid packing density and, consequently, reduces permeation rates.

Orientation and Dynamics of an Antimicrobial Peptide in the Lipid Bilayer by Solid-State NMR Spectroscopy

The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.

Dynamics of Membrane Penetration of the Fluorescent 7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl (NBD) Group Attached to an Acyl Chain of Phosphatidylcholine

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.

Characterization of the Ternary Mixture of Sphingomyelin, POPC, and Cholesterol: Support for an Inhomogeneous Lipid Distribution at High Temperatures

A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, (2)H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by (2)H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger (2)H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization.

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG–protein systems.

Membrane binding of lipidated Ras peptides and proteins — The structural point of view

Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.

The Distribution of Lipid Attached Spin Probes in Bilayers: Application to Membrane Protein Topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.

The Lipid Modifications of Ras that Sense Membrane Environments and Induce Local Enrichment

The transduction of an external stimulus from the outside of a cell into its nucleus is one of the most important mechanisms for the regulation of numerous biological processes. External signals activate receptors that transmit the information across the membrane, where it is transducted by a set of proteins that activate ion channels, phosphokinases, or other downstream effectors. GTP binding proteins that pick up the signal at the receptor, such as heterotrimeric G proteins or Ras, are membrane-associated by post-translationally acquired lipid modifications. These lipid chains provide the hydrophobic free energy for membrane association and their lack releases the proteins to the cytosol, rendering them inactive. Thus, through membrane binding, Ras increases its effective concentration to optimize the interaction both with the receptor and downstream effectors. Ras is an important molecular switch that regulates cell proliferation, differentiation, and growth. The highly specific membrane binding of Ras can be appreciated by comparing the members of the Ras family: Two lipid modifications are required for N-Ras and K-Ras4A, whereas H-Ras carries three lipid chains. In contrast, K-Ras4B requires the concerted action of one lipid chain and favorable electrostatics for membrane binding. Although inserted into the membrane, the lipid modifications experience a high degree of motional freedom that is also transmitted to the adjacent polypeptide chain. Although the highly homologous Ras proteins interact with the same effectors in vitro, they produce distinctly different output signals in vivo, which suggests that these differences are imparted by the lipid-modified C termini of the proteins, where the homology is very low. Moreover, depending on the nucleotide binding state, the localization of Ras in liquid-crystalline or raft domains of the membrane appears to be regulated. Only active H-Ras*GTP interacts with the respective set of effectors; the non-activated form, H-Ras*GDP, is constrained to rafts, where the signal is not further transmitted. An alternative model suggests that the difference in signaling of the Ras isoforms is imparted from the altered access and residence time in a specific compartment. 10] This model suggests that interactions of Ras and its lipid modifications with rafts or fluid membrane domains determines the membrane localization and the biological function of the molecule, which is investigated herein. H NMR is a useful tool for the investigation of lipid rafts. It is applicable to each component of a lipid mixture, and only requires the synthesis of the relevant molecule with a deuterated chain. First, we investigated the adaptation of the lipid modifications of a N-Ras heptapeptide, which was hexadecylated at Cys181 and Cys186, to the membrane thickness. Four different membranes composed of lipids with varying hydrocarbon chains were chosen to constitute the host membrane. Membrane thicknesses studied by H NMR varied from 21.0 (DLPC) to 38.8 (DPPC/cholesterol 10:6, Table 1). The high cholesterol content leads to condensation of the lipids, which increases their length and abolishes the phase transitions of DPPC such that all lipid mixtures could be studied at 30 8C.

Comparison of collagen dynamics in articular cartilage and isolated fibrils by solid-state NMR spectroscopy

Native pig articular cartilage was investigated by 13C cross polarization (CP) magic angle spinning (MAS) NMR at a magnetic field strength of 17.6 T. CP MAS spectra of cartilage are dominated by resonances from rigid collagen, while only low‐intensity signals from the glycosaminoglycans are observed. The spectral resolution of collagen fibrils in native cartilage is somewhat higher than for isolated collagen fibrils from bovine achilles tendon investigated for comparison. This is confirmed qualitatively by 1H‐1H wideline separation spectra that show much lower line widths for cartilage collagen compared to isolated collagen. The strength of 1H‐13C dipolar couplings was measured in a 2D LG CP experiment providing a motionally averaged dipolar coupling value for each resolved signal. These scaled couplings were converted to molecular order parameters for the CH bond vector. Typical order parameters for isolated collagen were 0.91–0.96 for sidechains and 0.98–1.00 for the backbone. Somewhat lower order parameters were determined for cartilage collagen; 0.79–0.90 for the sidechain and 0.92–0.97 for the backbone. The only glycosaminoglycan signals that could be detected by CP MAS show order parameters of 0.48–0.92 and are assigned to relatively rigid hyaluronan and keratan sulfate. The higher mobility of collagen in cartilage is due to the high water content and collisions with the isotropically mobile glycosaminoglycans, such as chondroitin sulfate. Therefore, the mobility of cartilage macromolecules is broadly distributed from almost completely rigid to highly mobile, which lends cartilage its mechanical strength and shock‐absorbing properties. Magn Reson Med 48:624–632, 2002.