Biomedical subjects

P Laggner

Publications and source records attributed to P Laggner.

At least 19 recordsLinked to original sources

PubMed2001

Mechanism of the interaction of beta(2)-glycoprotein I with negatively charged phospholipid membranes.

In an attempt to understand the multifunctional involvement of beta(2)-glycoprotein I (beta(2)GPI) in autoimmune diseases, thrombosis, atherosclerosis, and inflammatory processes, substantial interest is focused on the interaction of beta(2)GPI with negatively charged ligands, in particular, with acidic phospholipids. In this study, unilamellar vesicles composed of cardiolipin were used as in vitro membrane system to test and further refine a model of interaction based on the crystal structure of beta(2)GPI. The data suggest that beta(2)GPI anchors to the membrane surface with its hydrophobic loop adjacent to the positively charged lysine rich region in domain V. Subsequently, beta(2)GPI penetrates the membrane interfacial headgroup region as indicated by a restriction of the lipid side chain mobility, but without formation of a nonbilayer lipid phase. A structural rearrangement of beta(2)GPI upon lipid binding was detected by microcalorimetry and may result in the exposure of cryptic epitopes located in the complement control protein domains. This lipid-dependent conformational change may induce oligomerization of beta(2)GPI and promote intermolecular associations. Thus, the aggregation tendency of beta(2)GPI may serve as the basis for the formation of a molecular link between cells but may also be an essential feature for binding of autoantibodies and hence determine the role of beta(2)GPI in autoimmune diseases.

Apolipoproteins↗

PubMed2001

Differential membrane fluidization by active and inactive cannabinoid analogues.

The effects of the two cannabinomimetic drugs (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]pyranyl-2-(hexyl)-1,3-dithiolane (AMG-3) and its pharmacologically less active 1-methoxy analogue (AMG-18) on the thermotropic and structural properties of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) liposomes have been studied by X-ray diffraction and differential scanning calorimetry (DSC). DSC data revealed that the incorporation of the drugs affect differently the thermotropic properties of DPPC. The presence of the more active drug distinctly broadened and attenuated both the pretransition and main phase transition of DPPC bilayers, while the inactive analogue had only minor effects. Small and wide angle X-ray diffraction data showed that the two cannabinoids have different effects on the lipid phase structures and on the hydrocarbon chain packing. The pharmacologically active analogue, AMG-3, was found to efficiently fluidize domains of the lipids in the L(beta)' gel phase, and to perturb the regular multibilayer lattice. In the liquid crystalline L(alpha) phase, AMG-3 was also found to cause irregularities in packing, suggesting that the drug induces local curvature. At the same concentration, the inactive AMG-18 had only minor structural effects on the lipids. At about 10-fold or higher concentrations, AMG-18 was found to produce similar but still less pronounced effects in comparison to those observed by AMG-3. The dose-dependent, different thermotropic and structural effects by the two cannabinoid analogues suggest that these may be related to their biological activity.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteins in vitro.

In many biological membranes, the major lipids are "non-bilayer lipids," which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that the in vitro association of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a/b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.

Circular Dichroism↗

PubMed2000

New evidence for gel-liquid crystalline phase coexistence in the ripple phase of phosphatidylcholines.

Experimental evidence supporting the hypothesis of gel-liquid crystalline phase coexistence in the stable ripple phase of diacylphosphatidylcholines has been obtained from time-resolved X-ray small- (SAXS) and wide-angle diffraction (WAXS) in the millisecond to second time domain. The pretransition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) exhibits a thin lamellar liquid crystalline intermediate phase (designated Lalpha) if driven far away from equilibrium by an infrared temperature jump (T-jump) technique. The findings can be described by a two-step model. (1) Instantaneously with the T-jump, an anomalously thin lamellar liquid crystalline intermediate phase (d = 5.6-5.8 nm) forms, coexisting with the original gel-phase Lbeta'. Within the first seconds, the lamellar repeat distance of the intermediate increases to a value of about 6.7 nm. A closer examination of these kinetics reveals two relaxation components: a fast process, proceeding within tenths of a second, and a slow process, on the time scale of a few seconds. (2) Finally, both the liquid crystalline and the gel-phase relax into the stable ripple phase Pbeta'. The total process time of the transition is nearly independent of the addition of NaCl, but varies strongly with the chain length of the lecithin species.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Self-regulation of the lipid content of membranes by non-bilayer lipids: a hypothesis.

Many biological membranes contain lipids that do not form a lamellar phase but the roles of these lipids are not well understood. An artificial membrane assembled from the main non-bilayer lipid and the major integral protein of pea thylakoids revealed that the protein spatially inhibits the formation of non-bilayer structures in the lamellae. Without this inhibition, excess lipids are secreted, creating lipid reservoirs for metabolism and/or later uptake. This determines the protein:lipid ratio in the membrane and hence the balance between structural flexibility and the stability of the key constituents that participate in cooperative interactions.

Lipid Bilayers↗

PubMed2000

Structural information from multilamellar liposomes at full hydration: full q-range fitting with high quality x-ray data.

We present a method for analyzing small angle x-ray scattering data on multilamellar phospholipid bilayer systems at full hydration. The method utilizes a modified Caillé theory structure factor in combination with a Gaussian model representation of the electron density profile such that it accounts also for the diffuse scattering between Bragg peaks. Thus the method can retrieve structural information even if only a few orders of diffraction are observed. We further introduce a procedure to derive fundamental parameters, such as area per lipid, membrane thickness, and number of water molecules per lipid, directly from the electron density profile without the need of additional volumetric measurements. The theoretical apparatus is applied to experimental data on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine liposome preparations.

Liposomes↗

PubMed1999

Crystal structure of human beta2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome.

The high affinity of human plasma beta2-glycoprotein I (beta(2)GPI), also known as apolipoprotein-H (ApoH), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. beta(2)GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the beta(2)GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central beta-spiral of four antiparallel beta-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313-316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The beta(2)GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of beta(2)GPI.

Amino Acid Sequence↗

PubMed1999

Microphase separation in low density lipoproteins. Evidence for a fluid triglyceride core below the lipid melting transition.

The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1. 0244-1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 degreesC, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core.

Calorimetry, Differential Scanning↗

PubMed1998

Crystallization and preliminary X-ray crystallographic studies on apolipoprotein H (beta2-glycoprotein-I) from human plasma.

Apolipoprotein-H (Apo-H, Mw approximately 50 kDa) is a carbohydrate-rich human-plasma protein which exists in blood serum in the free form as well as distributed between several classes of lipoproteins. Single crystals of apo-H have been obtained and crystallographic data sets have been collected. The crystals belong to the orthorhombic space group C2221, with cell dimensions a = 158. 47, b = 169.25, c = 113.28 A (at 100 K). The data indicate that the crystallographic asymmetric unit contains one tetramer of the protein.

Crystallization↗

PubMed1998

Lalpha-phase separation in phosphatidylcholine-water systems induced by alkali chlorides.

The effects of alkali chlorides on phosphatidylcholine-water bilayer systems in the Lalpha-phase were investigated by using small- and wide-angle X-ray scattering. The ternary system LiCl-POPC-H2O under isothermal conditions has shown that above Li+/POPC molar ratios of 0.1 and a lipid concentration above 5% (w/w), a splitting of the lamellar Bragg diffraction peaks into discrete components indicates a phase separation into different lamellar liquid crystalline (smectic A) phases. It is also shown that in saturated distearoyl phosphatidylcholine and in egg phosphatidylcholine, alkali chlorides induce Lalpha-phase separation. The number and repeat distance of the coexisting lamellar phases depend on the nature and concentration of the alkali chloride, the concentration of the phosphatidylcholine, and the degree of the acyl chain unsaturation.

Chemical Phenomena↗

PubMed1998

Thermal stability of apolipoprotein B100 in low-density lipoprotein is disrupted at early stages of oxidation while neutral lipid core organization is conserved.

The time course of the unfolding characteristics of the protein moiety and of the thermotropic behavior of the core-located apolar lipids of highly homogeneous low-density lipoprotein (LDL) subspecies (d 1.030-1.040 g/mL) have been evaluated during transition metal- and azo radical-induced oxidation using differential scanning calorimetry. Apolipoprotein B100 (apo-B100) structure was highly sensitive to oxidative modification; indeed, a significant loss of thermal stability was observed at initial stages irrespective of whether oxidation was mediated by site-specific binding of copper ions or by free radicals generated during decomposition of azo compounds. Subsequently, thermal protein integrity was destroyed, as a result of potentially irreversible protein unfolding, cross-linking reactions, and aggregation. Our results suggest that even minimal oxidative modification of apo-B100 has a major impact on the stability of this large monomeric protein. By contrast, the core lipids, which consist primarily of cholesteryl esters and triglycerides and play a determinant role in the thermal transition occurring near physiological temperature, preserved features of an ordered arrangement even during propagation of lipid peroxidation.

Amidines↗

PubMed1998

Surface Roughness Created by Acidic Dissolution of Synthetic Goethite Monitored with SAXS and N2-Adsorption Isotherms

The development of surface roughness of two synthetic goethites with a high, 176 m2/g, and a low, 7 m2/g, initial specific surface area by acid attack is described in terms of fractal geometry. The dissolution kinetics were influenced by the presence of micropores and small mesopores created during dissolution and leading to an initial increase in surface area causing S-shaped dissolution curves. The fractal dimensions were calculated from the adsorption isotherms obtained with N2 as adsorbate. These values of surface fractal dimension agree quite well with the values of the mass and surface fractal dimension obtained with small angle X-ray scattering (SAXS). Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗

PubMed1998

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

Adenosine Triphosphate↗

PubMed1998

Site-specific effect of radical scavengers on the resistance of low density lipoprotein to copper-mediated oxidative stress: influence of alpha-tocopherol and temperature.

The radical scavenging capacity of active nitroxide spin label radicals located at different depths in the surface monolayer of native and alpha-tocopherol enriched low density lipoprotein (LDL) has been evaluated at early stages of copper-mediated lipid peroxidation. Spin labels induced a concentration-dependent prolongation in lag time and a pronounced decrease in the initial rate of conjugated diene (CD) formation. These effects strongly argue for a protective, antioxidative action of spin labels, which in turn become destroyed with the extent of oxidation by radical recombination reactions. The results revealed that the decrease in spectral intensity proceeds at a higher rate for nitroxide radicals located in a more hydrophobic environment. The loss in spin label activity is accompanied by simultaneous alpha-tocopherol consumption and progresses rather independently of initial alpha-tocopherol content. The data provided no evidence that spin labels either save alpha-tocopherol or compete with it for radicals. The authors, therefore, deduce that due to enhanced accessibility and mobility, spin labels located in the interior of LDL eliminate lipid-derived radicals, which otherwise would promote lipid peroxidation. Lowering of temperature clearly below the core-lipid phase transition temperature of LDL exerts a significant effect on the kinetics of copper-induced LDL oxidation, whereas the characteristics of the radical scavenging mechanisms of the spin label molecules located in the surrounding phospholipid monolayer are conserved. Taken together, the susceptibility of LDL to primary oxidative stress conditions was efficiently retarded by small amounts of radical scavengers. This effect was more pronounced for nitroxide radicals embedded deeper in the phospholipid monolayer and was rather independent of alpha-tocopherol enrichment.

Copper↗

PubMed1998

Trapping of short-lived intermediates in phospholipid phase transitions: the L* alpha phase.

Time-resolved small-angle X-ray diffraction of liquid-crystalline phospholipid-water systems under temperature or pressure jump conditions has demonstrated the existence of an ordered, intermediate L alpha phase, with a sub-second lifetime, designated as the L* alpha-phase. The lamellar repeat spacing is, universally, 0.3 nm smaller than that of the parent phase, irrespective of the lipid composition and of the jump conditions, provided that the jump leads to a net volume expansion of the phase. The presence of salts, most notably LiCl, leads to a prolongation of the lifetime. The results suggest a non-monotonic potential function for the interbilayer water thickness.

Membranes, Artificial↗

PubMed1997

Characterization of the sub-main-transition in distearoylphosphatidylcholine studied by simultaneous small- and wide-angle X-ray diffraction.

Simultaneous small- and wide-angle X-ray diffraction was used to investigate the structural conversions in the so-called sub-main-transition of fully hydrated multilamellar vesicles of distearoyl phosphatidylcholine (DSPC). The small-angle diffraction patterns show a modification in the supramolecular structure of the corrugated lipid bilayer and reveal that the sub-main-transition does not abolish the general features of the ripple phase (P(beta')). Concomitantly in the wide-angle region the diffraction patterns exhibit a rearrangement of the hydrocarbon chain packing and an onset of chain melting. The presence of KCl show an enhancement of the effects of the sub-main-transition. Moreover, in the ripple phase (P(beta')) below the sub-main-transition the hydrocarbon chains are positioned on an orthorhombic lattice in the presence of KCl.

Calorimetry, Differential Scanning↗

PubMed1996

Crystallization and preliminary X-ray analysis of a low density lipoprotein from human plasma.

Single crystals of human plasma low density lipoprotein (LDL), the major transport vehicle for cholesterol in blood, have been produced with a view to analysis of the three-dimensional structure by x-ray crystallography. Crystals with dimensions of approximately 200 x 100 x 50 microm have been reproducibly obtained from highly homogeneous LDL particle subspecies, isolated in the density ranges d = 1.0271-1. 0297 g/ml and d = 1.0297-1.0327 g/ml. Electron microscopic imaging of ultrathin-sectioned preparations of the crystals confirmed the existence of a regular, quasihexagonal arrangement of spherical particles of approximately 18 nm in diameter, thereby resembling the dimensions characteristic of LDL after dehydration and fixation. X-ray diffraction with synchrotron radiation under cryogenic conditions revealed the presence of well resolved diffraction spots, to a resolution of about 29 A. The diffraction patterns are indexed in terms of a triclinic lattice with unit cell dimensions of a = 16. 1 nm, b = 39.0 nm, c = 43.9 nm; alpha = 96.2 degrees, beta = 92.1 degrees, gamma = 102 degrees, and with space group P1.

Crystallography, X-Ray↗

PubMed1995

Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.

Calorimetry, Differential Scanning↗