Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-State NMR

The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.     

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

The orientation of cytochromes in membrane multilayers prepared from aerobically grown Escherichia coli K12

Centrifugation of membrane vesicles, prepared from ultrasonically disrupted Escherichia coli K12, on to a planar surface followed by slow, partial dehydration results in a high degree of parallel orientation of the membrane planes with respect to each other and the supporting surface. Rotation of such membrane multilayers about a single axis parallel with the membrane planes within the magnetic field of an electron paramagnetic resonance (e.p.r.) spectrometer allows the orientation of anisotropic paramagnetic centres to be deduced. Computer simulations of the angular dependence of cytochrome e.p.r. spectra show two, or perhaps three, cytochromes, well-oriented with respect to the membrane plane. A low-spin cytochrome is oriented with the normal to its haem plane lying in the membrane plane. One (or perhaps two) high-spin cytochrome(s) lies with its haem plane making an angle of 45 degrees with the membrane plane. The orientation of the low-spin cytochrome haem is thus the same as that of haems in b-type cytochromes and cytochrome oxidases of the a type found in the mitochondria of higher animal and microbial cells and the bacterium Paracoccus denitrificans (Erecińska et al., 1979). The possible identity of this low-spin component as the terminal oxidase, cytochrome o, is discussed.     

Effects of cannabinoids in membrane bilayers containing cholesterol.

The thermotropic and dynamic properties of the biologically active Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and its inactive congener O-methyl-Delta(8)-tetrahydrocannabinol (Me-Delta(8)-THC) in DPPC/cholesterol (CHOL) bilayers have been studied using a combination of DSC and solid-state NMR spectroscopy. The obtained results showed differential effects of the two cannabinoids under study. These are summarized as follows: (a) the presence of the active compound fluidizes more significantly the DPPC/CHOL bilayers than the inactive analog as it is revealed by DSC and NMR spectroscopy results; (b) cholesterol seems to play a significant role in the way cannabinoids act in membrane bilayers; (c) the observed additional peaks in (13)C/MAS-NMR spectra which were cannabinoid specific offer an evidence of their different dynamic properties in membranes. In particular, the aromatic part of the inactive cannabinoid appears more mobile than that of the active one. This finding is in agreement with previously obtained X-ray data which locate the inactive cannabinoid in the hydrophobic core of the bilayer while the active one in the polar region; and (d) the observed downfield shift of C-1 carbon in the preparation containing the active cannabinoid is a strong evidence that Delta(8)-THC resides nearby the polar region where also cholesterol is well known to locate itself. Such downfield shift is absent when Me-Delta(8)-THC is resided in the membrane bilayer. These differential effects of the two cannabinoids propose that the phospholipid/cholesterol core of the membrane may play an important role in the mode of cannabinoid action by regulating their thermotropic and dynamic properties.     

Orientation of the tyrosyl D, pheophytin anion, and semiquinone Q(A)(*)(-) radicals in photosystem II determined by high-field electron paramagnetic resonance

The radical forms of two cofactors and an amino acid in the photosystem II (PS II) reaction center were studied by using high-field EPR both in frozen solution and in oriented multilayers. Their orientation with respect to the membrane was determined by using one-dimensionally oriented samples. The ring plane of the stable tyrosyl radical, Y(D)(*), makes an angle of 64 degrees +/- 5 degrees with the membrane plane, and the C-O direction is tilted by 72 degrees +/- 5 degrees in the plane of the radical compared to the membrane plane. The semiquinone, Q(A)(*)(-), generated by chemical reduction in samples lacking the non-heme iron, has its ring plane at an angle of 72 degrees +/- 5 degrees to the membrane plane, and the O-O axis is tilted by 21 degrees +/- 5 degrees in the plane of the quinone compared to the membrane plane. This orientation is similar to that of Q(A)(*)(-) in purple bacteria reaction centers except for the tilt angle which is slightly bigger. The pheophytin anion was generated by photoaccumulation under reducing conditions. Its ring plane is almost perpendicular to the membrane with an angle of 70 degrees +/- 5 degrees with respect to the membrane plane. This is very similar to the orientation of the pheophytin in purple bacteria reaction centers. The position of the g tensor with respect to the molecule is tentatively assigned for the anion radical on the basis of this comparison. In this work, the treatment of orientation data from EPR spectroscopy applied to one-dimensionally oriented multilayers is examined in detail, and improvements over previous approaches are given.     

Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR

The orientation of a beta-sheet membrane peptide in lipid bilayers is determined, for the first time, using two-dimensional (2D) (15)N solid-state NMR. Retrocyclin-2 is a disulfide-stabilized cyclic beta-hairpin peptide with antibacterial and antiviral activities. We used 2D separated local field spectroscopy correlating (15)N-(1)H dipolar coupling with (15)N chemical shift to determine the orientation of multiply (15)N-labeled retrocyclin-2 in uniaxially aligned phosphocholine bilayers. Calculated 2D spectra exhibit characteristic resonance patterns that are sensitive to both the tilt of the beta-strand axis and the rotation of the beta-sheet plane from the bilayer normal and that yield resonance assignment without the need for singly labeled samples. Retrocyclin-2 adopts a transmembrane orientation in dilauroylphosphatidylcholine bilayers, with the strand axis tilted at 20 degrees +/- 10 degrees from the bilayer normal, but changes to a more in-plane orientation in thicker 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) bilayers with a tilt angle of 65 degrees +/- 15 degrees . These indicate that hydrophobic mismatch regulates the peptide orientation. The 2D spectra are sensitive not only to the peptide orientation but also to its backbone (phi, psi) angles. Neither a bent hairpin conformation, which is populated in solution, nor an ideal beta-hairpin with uniform (phi, psi) angles and coplanar strands, agrees with the experimental spectrum. Thus, membrane binding orders the retrocyclin conformation by reducing the beta-sheet curvature but does not make it ideal. (31)P NMR spectra of lipid bilayers with different compositions indicate that retrocyclin-2 selectively disrupts the orientational order of anionic membranes while leaving zwitteronic membranes intact. These structural results provide insights into the mechanism of action of this beta-hairpin antimicrobial peptide.     

WITHDRAWN: Heme oxygenase-1 mediates the anti-inflammatory effect of propyl gallate in LPS-stimulated macrophages

Tryptophans in membrane proteins display strong preference for the lipid membrane interface and are important for anchoring proteins at the proper longitudinal level. Linear dichroism spectroscopy on indoles in shear-deformed liposomes has been used to show that this positioning is accompanied by an intrinsically adopted orientation, also observed for tryptophans in membrane-bound peptides. Similarities in orientation of different indoles suggest that tryptophan will adopt this orientation independent of the protein it is part of. From the orientation of indole electronic transition moments L(a), L(b) and B(b), a binding model is proposed where the indole long axis is approximately 60-65 degrees from the membrane normal and the indole plane is at an oblique angle. We propose that dipole-dipole interactions and steric constraints in the membrane hydrocarbon region determine positioning and orientation of tryptophans whereas hydrogen bonding and cation-pi interactions with lipid head-groups, though contributing to the membrane affinity of indoles, are less important.     

How lipophilic cannabinergic ligands reach their receptor sites

It is postulated that lipophilic ligands reach their sites of action on membrane-bound functional proteins through fast lateral diffusion across the membrane bilayer. We have shown using NMR experiments that such ligands when incorporated in a membrane system assume a preferred orientation and conformation. While occupying a specific location within the bilayer, these molecules undergo fast lateral diffusion which allows them to engage in productive interactions with their respective protein sites of action. The proposed model is discussed using a group of classical and non-classical cannabinoids as well as the endogenous cannabinoid ligand anandamide.     

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (g_z) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. the tetranuclear iron-sulfur clusters can have their g_z axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment

The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation.     

Molecular organization of 2-(2,4-dihydroxylphenyl)-5,6-dichlor 1,3-benzothiazole in monomolecular layers formed with diphytanoylphosphatidylcholine: A linear dichroism-FTIR study

2-(2,4-Dihydroxylphenyl)-5,6-dichlor 1,3-benzothiazole (dHBBT) has very strong antifungal and antitumoral properties in relation to human cancer cells. The aim of this research was to analyze the binding process of dHBBT molecules to the lipid membrane formed from DPhPC at the air-water interface. The effect of dHBBT on the organization of lipid membranes formed with diphytanoylphosphatidylcholine (DPhPC) was studied with the application of monomolecular layer technique, FTIR spectroscopy and linear dichroism-FTIR. On the basis of linear dichroism experiments the mean orientation angle θ between the molecular axis and the normal to monolayer surface was determined. Mean value was calculated at θ = 72° and indicates a horizontal orientation of dHBBT molecules in the lipid membrane formed from DPhPC. dHBBT molecules have considerable influence on the orientation of DPhPC acyl chains. The mean value of the angle between normal to monolayer surface and the main axis of the acyl chain is approximately 45° for DPhPC, while for the lipid monolayer containing dHBBT it is approximately 18°. Such extreme changes in orientation of acyl chains indicate a clear influence of the relationship on the dynamic and structural properties of the monolayer formed from DPhPC. Biological activity of dHBBT molecules is tightly associated with its molecular organization. The results of the research presented in this work are potentially valuable in respect of the development of pharmacologically active preparations of dHBBT.     

Contextual Effects of Scene on the Visual Perception of Object Orientation in Depth

We investigated the effect of background scene on the human visual perception of depth orientation (i.e., azimuth angle) of three-dimensional common objects. Participants evaluated the depth orientation of objects. The objects were surrounded by scenes with an apparent axis of the global reference frame, such as a sidewalk scene. When a scene axis was slightly misaligned with the gaze line, object orientation perception was biased, as if the gaze line had been assimilated into the scene axis (Experiment 1). When the scene axis was slightly misaligned with the object, evaluated object orientation was biased, as if it had been assimilated into the scene axis (Experiment 2). This assimilation may be due to confusion between the orientation of the scene and object axes (Experiment 3). Thus, the global reference frame may influence object orientation perception when its orientation is similar to that of the gaze-line or object.     

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the beta-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant alpha-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular 31P-1H and 1H-1H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.     

The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol

Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-side-down" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.     

The conformation, location, and dynamic properties of the endocannabinoid ligand anandamide in a membrane bilayer

The endogenous cannabinoid ligand anandamide is biosynthesized from membrane phospholipid precursors and is believed to reach its sites of action on the CB1 and CB2 receptors through fast lateral diffusion within the cell membrane. To gain a better insight on the stereochemical features of its association with the cell membrane and its interaction with the cannabinoid receptors, we have studied its conformation, location, and dynamic properties in a dipalmitoylphosphatidylcholine multilamellar model membrane bilayer system. By exploiting the bilayer lattice as an internal three-dimensional reference grid, the conformation and location of anandamide were determined by measuring selected inter- and intramolecular distances between strategically introduced isotopic labels using the rotational echo double resonance (REDOR) NMR method. A molecular model was proposed to represent the structural features of our anandamide/lipid system and was subsequently used in calculating the multispin dephasing curves. Our results demonstrate that anandamide adopts an extended conformation within the membrane with its headgroup at the level of the phospholipid polar group and its terminal methyl group near the bilayer center. Parallel static (2)H NMR experiments further confirmed these findings and provided evidence that anandamide experiences dynamic properties similar to those of the membrane phospholipids and produces no perturbation to the bilayer. Our results are congruent with a hypothesis that anandamide approaches its binding site by laterally diffusing within one membrane leaflet in an extended conformation and interacts with a hydrophobic groove formed by helices 3 and 6 of CB1, where its terminal carbon is positioned close to a key cysteine residue in helix 6 leading to receptor activation.     

Correlation of nuclear acceptor sites for oestrogen receptors with gene transcription in vitro

1. Membrane particles prepared from ultrasonically-disrupted, aerobically-grown Escherichia coli were centrifuged on to a plastic film that was supported perpendicular to the centrifugal field to yield oriented membrane multilayers. In such preparations, there is a high degree of orientation of the planes of the membranes such that they lie parallel to each other and to the supporting film. 2. When dithionite- or succinate-reduced multilayers are rotated in the magnetic field of an e.p.r. spectrometer, about an axis lying in the membrane plane, angular-dependent signals from an iron-sulphur cluster at g(x)=1.92, g(y)=1.93 and g(z)=2.02 are seen. The g=1.93 signal has maximal amplitude when the plane of the multilayer is perpendicular to the magnetic field. Conversely, the g=2.02 signal is maximal when the plane of the multilayer is parallel with the magnetic field. 3. Computer simulations of the experimental data show that the cluster lies in the cytoplasmic membrane with the g(y) axis perpendicular to the membrane plane and with the g(x) and g(z) axes lying in the membrane plane. 4. In partially-oxidized multilayers, a signal resembling the mitochondrial high-potential iron-sulphur protein (Hipip) is seen whose g(z)=2.02 axis may be deduced as lying perpendicular to the membrane plane. 5. Appropriate choice of sample temperature and receiver gain reveals two further signals in partially-reduced multilayers: a g=2.09 signal arises from a cluster with its g(z) axis in the membrane plane, whereas a g=2.04 signal is from a cluster with the g(z) axis lying along the membrane normal. 6. Membrane particles from a glucose-grown, haem-deficient mutant contain dramatically-lowered levels of cytochromes and exhibit, in addition to the iron-sulphur clusters seen in the parental strain, a major signal at g=1.90. 7. Only the latter may be demonstrated to be oriented in multilayer preparations from the mutant. 8. Comparisons are drawn between the orientations of the iron-sulphur proteins in the cytoplasmic membrane of E. coli and those in mitochondrial membranes. The effects of diminished cytochrome content on the properties of the iron-sulphur proteins are discussed.     

Tryptophan orientations in membrane-bound gramicidin and melittin-a comparative linear dichroism study on transmembrane and surface-bound peptides

In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the non-channel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions.     

Topo-optical study of the pulmonary ciliary membrane surface.

In hydrophilic media of high refraction index the cilia of the epithelium of the tracheo-bronchial mucous membrane and the mucous film demonstrate birefringence positive to the longitudinal axis of the mucous membrane owing to the identical orientation the glycoprotein macromolecules and of the fatty acid chains of the ciliary membrane. By means of toluidine blue staining the mucous film and the ciliary zone can be studied selectively by polarization microscopy. The opposite optical character suggests that the dye molecules are perpendicularly connected to the long glycoprotein chains of the mucous, and there is an oriented connection perpendicular to the longitudinal axis of the cilium in its lipid membrane. In smears of bronchial secretion or mucous membrane scrapings after staining, with toluidine blue pH 7.0, the selective optical reaction of the cilia provides a possibility for the study of isolated desquamated ciliary cells. The reaction is given by the lipid membrane boundary of the cilia. In embedded, lipid-extracted sections, the cilia and the mucous film show a minimum of birefringence in the unstained state. After toluidine blue staining or aldehyde-bisulphite-toluidine blue (ABT) reaction the mucoid surface and the ciliary zone display an opposite optical reaction originating from the dehydrated mucoproteins adsorbed onto the surface of lipid-extracted cilia.