Peter Laggner

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.

Crystal structure of human β2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome

The high affinity of human plasma β2‐glycoprotein I (β2GPI), 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. β2GPI 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 β2GPI 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 β‐spiral of four antiparallel β‐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 β2GPI 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 β2GPI.

First performance assessment of the small-angle X-ray scattering beamline at ELETTRA

The double-focusing high-flux wiggler beamline dedicated to small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) at ELETTRA has gone into user operation recently. It has been designed specifically for time-resolved studies of non-crystalline and fibrous materials in the submillisecond time scale, and has been optimized for small-angle scattering measurements. An overview of the beamline status and of some representative results, highlighting the performance of the SAXS beamline, are given.

Structure and thermodynamics of the dihexadecylphosphatidylcholine-water system

X-ray small- and wide-angle diffraction, differential scanning calorimetry (DSC), temperature scanning densitometry (TSD) and electron microscopy were used to study the lyotropic and thermotropic properties of the system 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine-water over a wide range of compositions from the dry lipids to a large excess of water, and in the temperature range between 0 degrees C and 150 degrees C. The results were used to construct a temperature-composition phase diagram. The phases have been characterized with respect to their molecular arrangements and hydrocarbon chain packing subcells. In the fully hydrated state (greater than 45 wt% H2O) four thermotropic phases were found, with readily reversible transitions at 5 degrees C, 32.5 degrees C and 43.6 degrees C, respectively. The two lower temperature phases deviate from all others in consisting of bilayers with fully interdigitated hydrocarbon chains, while above 32.5 degrees C the structures resemble closely those of the analog diester lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). At hydrations between 30 and 45 wt% H2O, and below 32 degrees C, interdigitated and non-interdigitated multilayers coexist in one coherent phase. A bilayer tilting mechanism is proposed for the formation of this coexistence of two regular structures. Below 30 wt% H2O, hydrated 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) exists in lamellar, non-interdigitated bilayers, showing very weak interbilayer swelling. There, the water molecules appear to occupy voids between the polar headgroups.

Structural analysis of weakly ordered membrane stacks

The applicability of full-q-range models to fit low-resolution X-ray diffraction data from multibilayers exhibiting only weak quasi-Bragg peak scattering has been analysed. The models consider different structure factors, accounting for different types of lattice disorder caused by stacking faults or bending fluctuations. Numerical tests of the models, considering instrumental influence of a line-focus collimation system, demonstrated that Bragg peak line shapes given by different lattice disorders cannot be discerned. However, line-shape parameters can be determined for a particular sample, if the type of disorder is known a priori. This has been verified by comparing the experimental results for the fluctuation parameter of palmitoyl-oleoyl phosphatidylcholine (POPC) as a function of temperature with high-resolution data on the same lipid. Tests further show that the calculation of structural parameters, such as the membrane thickness or the extent of the interbilayer water region, is not obscured by the smearing imposed by the instrument. The model was further applied successfully to experimental data of lipid mixtures composed of sphingomyelin (SM)/POPC/cholesterol and dipalmitoyl phosphatidylethanolamine (DPPE)/dipalmitoyl phosphatidylglycerol (DPPG). The structural parameters determined give valuable insight into the physical state of the membrane system, which is not accessible when quasi-Bragg reflections only are considered.

High‐flux beamline for small‐angle x‐ray scattering at ELETTRA

The optical layout and the expected performance of the new high‐flux SAXS beamline at ELETTRA is presented. From the high‐power wiggler spectrum the three discrete energies 5.4, 8, and 16 keV will be selected with a double‐crystal monochromator which contains three pairs of separated asymmetrically cut plane Si(111) crystals. Downstream, the beam will be focused by a torodial mirror. The optical axis of the beamline will be horizontally 1.25 mrad off wiggler axis and the beamline will accept about 1 mrad horizontally and 0.3 mrad vertically. The beamline will operate with a SAXS resolution between 10 and at least 1000 A in d spacing at 8 keV and has been optimized with respect to extreme flux. A flux at the sample in the order of 1013 ph/s is expected for 8 keV photons (2 GeV, 400 mA). It will be possible to perform wide angle scattering measurements in the range of 3.5 and 7 A d spacing at 8 keV simultaneously.

Differential Modulation of Membrane Structure and Fluctuations by Plant Sterols and Cholesterol

We have studied the concentration and temperature dependent influence of cholesterol, stigmasterol, and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl phosphatidylcholine and palmitoyl oleoyl phosphatidylcholine bilayers applying small-angle x-ray scattering, as well as dilatometry and ultrasound velocimetry. Independent of the lipid matrix, cholesterol was found to be most efficient in modulating bilayer thickness and elasticity, followed by sitosterol and stigmasterol. This can be attributed to the additional ethyl groups and double bond at the C(17) alkyl side-chain of the two plant sterols. Hence, it seems that some flexibility of the sterol hydrocarbon chain is needed to accommodate within the lipid bilayer. In addition, we did not observe two populations of membranes within the putative liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. Instead, the diffraction patterns could be interpreted in terms of a uniform phase. This lends further support to the idea of compositional fluctuations of unstable sterol rich domains recently brought up by fluorescence microscopy experiments, which contrasts the formation of stable domains within the miscibility gap of binary lipid/sterol mixtures.

Performance and First Results of the ELETTRA High‐Flux Beamline for Small‐Angle X‐ray Scattering

A new beamline for small-angle X-ray scattering (SAXS) has recently been constructed and is presently under final commissioning at the 2 GeV storage ring ELETTRA. It has been designed specifically for time-resolved studies of non-crystalline and fibrous materials and has been optimized for small-angle scattering measurements. The beamline operates with a SAXS resolution between 10 and about 1400 A in d spacing (at 8 keV) and has been optimized with respect to high flux at the sample [of the order of 1013 photons s−1 for 8 keV photons (2 GeV, 400 mA)]. Soon it will be possible to perform simultaneously wide-angle diffraction measurements in the d-spacing range 1.2–8 A (at 8 keV). In order to allow time-resolved (resolution ~1 ms) small-angle scattering measurements, a high-power 57-pole wiggler is used as the beamline source. From its beam, one of three discrete energies, 5.4, 8 and 16 keV, can be selected with a double-crystal monochromator, which contains three pairs of asymmetrically cut plane Si(111) crystals. Downstream, the beam is focused horizontally and vertically by a toroidal mirror. Commissioning tests of this new SAXS beamline showed that all design parameters have been realized.

Tuning curvature and stability of monoolein bilayers by designer lipid-like peptide surfactants.

This study reports the effect of loading four different charged designer lipid-like short anionic and cationic peptide surfactants on the fully hydrated monoolein (MO)-based Pn3m phase (Q224). The studied peptide surfactants comprise seven amino acid residues, namely A6D, DA6, A6K, and KA6. D (aspartic acid) bears two negative charges, K (lysine) bears one positive charge, and A (alanine) constitutes the hydrophobic tail. To elucidate the impact of these peptide surfactants, the ternary MO/peptide/water system has been investigated using small-angle X-ray scattering (SAXS), within a certain range of peptide concentrations (R≤0.2) and temperatures (25 to 70°C). We demonstrate that the bilayer curvature and the stability are modulated by: i) the peptide/lipid molar ratio, ii) the peptide molecular structure (the degree of hydrophobicity, the type of the hydrophilic amino acid, and the headgroup location), and iii) the temperature. The anionic peptide surfactants, A6D and DA6, exhibit the strongest surface activity. At low peptide concentrations (R = 0.01), the Pn3m structure is still preserved, but its lattice increases due to the strong electrostatic repulsion between the negatively charged peptide molecules, which are incorporated into the interface. This means that the anionic peptides have the effect of enlarging the water channels and thus they serve to enhance the accommodation of positively charged water-soluble active molecules in the Pn3m phase. At higher peptide concentration (R = 0.10), the lipid bilayers are destabilized and the structural transition from the Pn3m to the inverted hexagonal phase (H2) is induced. For the cationic peptides, our study illustrates how even minor modifications, such as changing the location of the headgroup (A6K vs. KA6), affects significantly the peptides effectiveness. Only KA6 displays a propensity to promote the formation of H2, which suggests that KA6 molecules have a higher degree of incorporation in the interface than those of A6K.

Molecular structure of low density lipoprotein: current status and future challenges

This review highlights recent advances in structural studies on low density lipoprotein (LDL) with particular emphasis on the apolipoprotein moiety of LDL, apolipoprotein B100 (apoB100). Various molecular aspects of LDL are outlined and obstacles to structure determination are addressed. In this context, the prevailing conceptions of the molecular assembly of LDL and how the synergy of complementary biochemical, biophysical and molecular simulation approaches has lead to the current structural model of LDL are discussed. Evidence is presented that structural heterogeneity and the intrinsic dynamics of LDL are key determinants of the functionality of LDL in both health and disease. Some key research directions, remaining open questions and rapidly emerging new concepts for medical applications of LDL, are furthermore outlined. The article concludes by providing an outlook concerning promising future strategies for the clarification of the molecular details of LDL, in particular of apoB100, combining recent advances in molecular modeling with developments of novel experimental techniques. Although new insights into the molecular organization of LDL are forthcoming, many open questions remain unanswered. The major challenge of the next decade will certainly be the elucidation of the molecular structural and dynamic features of apoB100.