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.     

Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

The orientation of the reaction center bacteriochlorophyll dimer, (BChl)₂, and primary quinone, Q_I, has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl)₂ triplet and Q_I?Fe²⁺ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl)₂ triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q_I?Fe²⁺ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum) lying parallel to the plane of the membrane surface. The orientation of Q_I was determined by the angular dependence of this signal in Fe²⁺-depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.     

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q_y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q_y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q_x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.     

Dynamic structure of bombolitin II bound to lipid bilayers as revealed by solid-state NMR and molecular-dynamics simulation

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state ³¹P and ¹³C NMR spectroscopy. ³¹P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (T_c) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the T_c. ¹³C NMR spectra of site-specifically ¹³C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers.     

EPR signals and orientation of cytochromes in the spinach chloroplast thylakoid membrane

The cytochromes in spinach chloroplasts were studied using EPR spectroscopy. In addition to the low-spin heme signals previously assigned, cytochrome f (g_z 3.51), high-potential cytochrome b-559 (g_z 3.08) and cytochrome b-559 converted to a low-potential form (g_z 2.94), a high-spin heme signal was induced by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). However, this signal cannot be due to cytochrome b-563 in its native form. The orientation of the cytochromes in the thylakoid membrane was studied in magnetically oriented chloroplasts. Cytochrome b-559 in the native state and in the low-potential form was found to have its heme plane perpendicular to the membrane plane. The orientation was the same for cytochrome b-559 oxidized by low-temperature illumination, which suggests that also the reduced heme is oriented perpendicular to the membrane.     

Adrenocorticotropic Hormone Suppresses Gonadotropin-Stimulated Estradiol Release from Zebrafish Ovarian Follicles

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E₂) secretion. ACTH neither affected cortisol nor unstimulated E₂ release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E₂ secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL⁻¹. This effect of ACTH on E₂ release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E₂ biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Q_y transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Q_y transition of BChl c and the -carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Q_y transition is close to the magic angle (55°).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - LD linear dichroism Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Long-Range Surface Plasmon Polaritons for Efficient Optical Coupling in AlGaN/GaN Quantum Well Infrared Photodetector

In this paper, the supermodes, long-range surface plasmon polaritons (LRSPPs), have been theoretically studied to enhance the optical coupling of AlGaN/GaN quantum well infrared photodetector (QWIP) based on gold–Si₃N₄ hybrid architecture. The electromagnetic field, energy flow, and current density are analyzed by finite element method (FEM). In time domain, the electric field component E _z and current density J _z perpendicular to the multi-quantum wells (MQWs) are symmetric and asymmetric distributions over the gold grating, respectively, which precisely prove the existence of LRSPPs. The averaged |E _z |² across the whole quantum well region reaches 1.51?(V/m)² when the electric field intensity (|E ₀|²) of normal incidence is 1?(V/m)² at 4.65 μm. Extraordinarily low loss of the LRSPPs results in a coupling efficiency enhancement ratio of 2.23 in AlGaN/GaN QWIP compared with that obtained via bare gold grating with different polarized sources, exhibiting great potential for application in the focal plane arrays.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

Bimodal Polarotropism of Vaucheria to Polarized Blue Light: Parallel Polarotropism at High Fluence Rate Corresponds to Negative Phototropism

Vaucheria (Xanthophyceae) exhibited cruciform polarotropism when they were grown under polarized white or blue light for several days. The coexistence of two groups of branches growing perpendicular and parallel to the electric vector (E-vector) resulted in cruciform polarotropic orientation. Such polarotropic bending was, however, not detected within 24 hr. As the fluence rate of polarized white or blue light increased, parallel orientation to the E-vector became dominant. Polarized red light produced exclusively perpendicular polarotropism. This shift in pattern was much obvious in V. terrestris sensu G?tz than V. sessllis and V. dichotoma. Since the photoperception is restricted to the tip of the apical dome and since this region receives maximum photons when the E-vector is at a right angle to the cell axis, Vaucheria becomes oriented normal to the E-vector as far as the fluence rate is optimum. The direction of growth is expected to change into parallel to the E-vector when the fluence rate is supraoptimum. The perpendicular (normal) and parallel polarotropism of Vaucheria, thus, correspond to positive and negative phototropism, respectively. Orientation of photoreceptor molecules is suggested to be predominantly parallel to the surface of the apical dome. Received 14 June 1999/ Accepted in revised form 19 November 1999     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Estradiol Reduces Susceptibility of CD4+ T Cells and Macrophages to HIV-Infection

The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-β-estradiol (E₂) and ethinyl estradiol (EE) in HIV-infection of CD4⁺ T-cells and macrophages. Purified CD4⁺ T-cells and monocyte-derived macrophages were generated in vitro from peripheral blood and infected with R5 and X4 viruses. Treatment of CD4⁺ T-cells and macrophages with E₂ prior to viral challenge reduced their susceptibility to HIV infection in a dose-dependent manner. Addition of E₂ 2 h after viral challenge however did not result in reduced infection. In contrast, EE reduced infection in macrophages to a lesser extent than E₂ and had no effect on CD4⁺ T-cell infection. Reduction of HIV-infection induced by E₂ in CD4⁺ T-cells was not due to CCR5 down-regulation, but was an entry-mediated mechanism since infection with VSV-G pseudotyped HIV was not modified by E₂. In macrophages, despite the lack of an effect of E₂ on CCR5 expression, E₂–treatment reduced viral entry 2 h after challenge and increased MIP-1β secretion. These results demonstrate the direct effect of E₂ on susceptibility of HIV-target cells to infection and indicate that inhibition of target cell infection involves cell-entry related mechanisms.     

X-ray diffraction analysis of the 3D organization of collagen fibrils in the wall of the dogfish egg case

The wall of the egg case of the dogfish,Scyliorhinus canicula, contains a network-forming collagen assembled into a regular three-dimensional (3D) structure. It accomplishes supportive, protective and filtering functions for the embryo contained within it. The collagen molecules in the egg case are organized into a body-centred unit cell of dimensions (mean ± s.d.) (11.6 ± 1.0) nm X (11.6 ± 1.0) nm X (81.6 ± 3.2) nm, which belongs to the I422 space group. At a higher hierarchical level, the collagen molecules assemble into parallel arrays of fibrils, ca. 100 nm in diameter, which aggregate to form laminae ca. 0.5 μm thick. These laminae are organized into a plywood-like structure and account for 98% of the thickness of the wall of the egg case. X-ray diffraction patterns of the wall of the egg case were taken along mutually perpendicular directions, one being perpendicular to the surface of the egg case. Three different kinds of diffraction pattern were observed. One of the patterns was characteristic of an X-ray direction perpendicular to the laminae in the egg case (along the x-direction). The two other patterns were obtained with the X-rays directed parallel to the plane of the laminae, either along the capsule long axis (z) or perpendicular to this (y). These two patterns were observed interchangeably in either of the x- or y-directions depending on the specimen. The diffraction patterns were analysed and interpreted taking into consideration the 3D electron microscope data of the egg case. The results confirm and extend previous findings from transmission electron microscopy and low-angle X-ray diffraction and they suggest that there is only one major type of ordered collagen arrangement in the wall of the egg case.     

Bicelles: A natural ‘molecular goniometer’ for structural,dynamical and topological studies of molecules in membranes

Major biological processes occur at the biological membrane. One of the great challenges is to understand the function of chemical or biological molecules inside the membrane; as well of those involved in membrane trafficking. This requires obtaining a complete picture of the in situ structure and dynamics as well as the topology and orientation of these molecules in the membrane lipid bilayer. These led to the creation of several innovative models of biological membranes in order to investigate the structure and dynamics of amphiphilic molecules, as well as integral membrane proteins having single or multiple transmembrane segments. Because the determination of the structure, dynamics and topology of molecules in membranes requires a macroscopic alignment of the system, a new membrane model called ‘bicelles’ that represents a crossover between lipid vesicles and classical micelles has become very popular due to its property of spontaneous self-orientation in magnetic fields. In addition, crucial factors involved in mimicking natural membranes, such as sample hydration, pH and salinity limits, are easy to control in bicelle systems. Bicelles are composed of mixtures of long chain (14–18 carbons) and short chain phospholipids (6–8 carbons) hydrated up to 98% with buffers and may adopt various morphologies depending on lipid composition, temperature and hydration. We have been developing bicelle systems under the form of nano-discs made of lipids with saturated or biphenyl-containing fatty acyl chains. Depending on the lipid nature, these membranous nano-discs may be macroscopically oriented with their normal perpendicular or parallel to the magnetic field, providing a natural ‘molecular goniometer’ for structural and topological studies, especially in the field of NMR. Bicelles can also be spun at the magic angle and lead to the 3D structural determination of molecules in membranes.     

The conformation of the Congo-red ligand bound to amyloid fibrils HET-s(218–289): a solid-state NMR study

We have previously shown that Congo red (CR) binds site specifically to amyloid fibrils formed by HET-s(218–289) with the long axis of the CR molecule almost parallel to the fibril axis. HADDOCK docking studies indicated that CR adopts a roughly planar conformation with the torsion angle ? characterizing the relative orientation of the two phenyl rings being a few degrees. In this study, we experimentally determine the torsion angle ? at the center of the CR molecule when bound to HET-s(218–289) amyloid fibrils using solid-state NMR tensor-correlation experiments. The method described here relies on the site-specific ¹³C labeling of CR and on the analysis of the two-dimensional magic-angle spinning tensor-correlation spectrum of ¹³C₂-CR. We determined the torsion angle ? to be 19°.     

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

Oxygenation of the 5-lipoxygenase product 5S-hydroxyeicosatetraenoic acid by cyclooxygenase-2 yields a bicyclic di-endoperoxide. The di-endoperoxide contains two peroxides spanning from carbons 9 to 11 and 8 to 12, and two hydroxyls at carbons 5 and 15 of arachidonic acid (Schneider C., et al. 2006. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2. J. Am. Chem. Soc. 128: 720). Here, we report that treatment of the di-endoperoxide with hematin or ferrous chloride results in cleavage of both peroxide O-O bonds and of the bonds between the carbons that carry the peroxide groups, producing the aldehydes 4-hydroxy-2E-nonenal (4-HNE), 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde (MDA). The hematin- and ferrous iron-catalyzed transformation of the di-endoperoxide proceeded with a similar yield of products as the cleavage of the prostaglandin endoperoxide PGH₂ to 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid and MDA. Chiral phase HPLC analysis of the 4-HNE cleavage product showed greater than 98% 4S and thus established the S configuration of the 15-carbon of the di-endoperoxide that had not previously been assigned. This transformation of the 5-lipoxygenase/cyclooxygenase-2 derived di-endoperoxide invokes the possibility of a novel pathway to formation of the classic lipid peroxidation products 4-HNE and MDA.     

The location,orientation and stoichiometry of the Rieske iron-sulfur cluster in membranes from Rhodopseudomonas sphaeroides

Neutral and negatively charged dysprosium complexes are able to enhance the spin relaxation rate of the Rieske iron-sulfur cluster only when added from the cytochrome c₂ side of the photosynthetic membrane, indicating that the Rieske cluster is asymmetrically placed in the membrane, nearer the cytochrome c₂ side. The g_z-axis of the Rieske cluster, taken to be the iron-iron axis of this binuclear cluster, lies in the membrane plane, as does the g_y-axis. Appropriately, the g_x-axis is orthogonal to the membrane plane. A comparison with a mammalian mitochondrial standard indicates that there are 0.65 ± 0.1 Rieske cluster per reaction center. This is in excellent agreement with previously determined estimates of the number of antimycin-binding sites, and binding sites for what is known phenomenologically as Q_Z, suggesting that there is one of each per ubiquinol-cytochrome c₂ oxidoreductase.     

Differences between Pacemaker and Nonpacemaker Neurons of Aplysia on Voltage Clamping

The responses of pacemaker and nonpacemaker Aplysia neurons to voltage clamp commands of less than 200 msec duration are essentially identical. For moderate depolarizing commands there is an early inward transient current followed by a late outward current and an outward tail current when the membrane is clamped back to resting potential. On long (1–2 sec) commands in pacemakers there is a marked sag in the late current and an inward tail current. E_tail, the potential of the membrane at which there is no net current flow under the conditions prevailing at the end of the clamp, shifts from about -9.0 mv on short commands to +5.0 mv on long commands. In contrast there is no marked sag of the late current or inward tail current on long commands in nonpacemakers, and E_tail is near -9.0 mv for both short and long commands. The current sag and shift in E_tail can be ascribed to a decreased conductance (presumably to K⁺) at the end of the long as compared to the short command in half of the pacemaker neurons. In the remaining cells the essential difference from nonpacemakers appears to be either a greater restricted extracellular space or a more active potential-dependent electrogenic Na⁺ pump in pacemakers.