Initial metal-metal bond breakage detected by fs X-ray scattering in the photolysis of Ru3(CO)12 in cyclohexane at 400 nm.

Using femtosecond resolution X-ray solution scattering at a free electron laser we were able to directly observe metal-metal bond cleavage upon photolysis at 400 nm of Ru3(CO)12, a prototype for the photochemistry of transition metal carbonyls. This leads to the known single intermediate Ru3(CO)11(µ-CO)*, with a bridging ligand (µCO) and where the asterisk indicates an open Ru3-ring. This loses a CO ligand on a picosecond time scale yielding a newly observed triple bridge intermediate, Ru3(CO)8(µ-CO)3*. This loses another CO ligand to form the previously observed Ru3(CO)10, which returns to Ru3(CO)12via the known single-bridge Ru3(CO)10(µ-CO). These results indicate that contrary to long standing hypotheses, metal-metal bond breakage is the only chemical reaction immediately following the photolysis of Ru3(CO)12 at 400 nm. Combined with previous picosecond resolution X-ray scattering data and time resolved infrared spectroscopy these results yield a new mechanism for the photolysis of Ru3(CO)12.

Elevated CD8 T cell responses in type 1 diabetes patients to a 13 amino acid coeliac-active peptide from α-gliadin.

Some type 1 diabetes (T1D) patients have been reported to exhibit T cell reactivity to wheat gluten. We tested the hypothesis that this T cell reactivity could be abolished by using prolyl-endopeptidase (PEP), an enzyme that cleaves peptide bonds after proline. Peripheral blood mononuclear cells (PBMCs) were isolated from T1D patients and healthy controls. PBMCs were stimulated with a peptic-tryptic digest of wheat gluten; a peptic-tryptic-PEP digest of wheat gluten; and a 13 amino acid peptide from wheat gluten. Fluorescent-labelled antibodies to CD3, CD4 and CD8 cell marker proteins were utilized to determine proliferative responses of CD3, CD4 and CD8 T cells. There were no significant differences in proliferative responses of CD3 or CD4 T cells to the wheat gluten antigens. A significantly higher proportion of CD8(+) T cells from T1D patients proliferated in the presence of the 13 amino acid peptide than when challenged with the peptic-tryptic or the peptic-tryptic-PEP digests of wheat gluten. PEP treatment had no significant effect on CD8 T cell reactivity to the peptic-trytic digest of wheat gluten. Our results suggest that wheat gluten-derived peptides, containing ≤ 13 amino acids, may evoke T cell responses in T1D patients.

Structural prerequisites for endotoxic activity in the Limulus test as compared to cytokine production in mononuclear cells.

The structural prerequisites for lipopolysaccharide (LPS) and its partial structures for the activation of the Limulus clotting cascade (Limulus amebocyte lysate [LAL] test) are described and compared with the corresponding requirements for the activation of human immune cells such as mononuclear cells. A necessary, but not sufficient, structural motif for this is the presence of the 4(')-phosphate-diglucosamine backbone recognition structure ('epitope') in lipid A. High activity is only expressed by assemblies of endotoxins, but this is largely independent of the type of supramolecular aggregate structure. A particular conformation of the epitope within the lipid A assembly must be present, which is influenced by addition of further saccharide units to the lipid A moiety, but also reacts slightly to the acylation pattern. In contrast, the cytokine production of human immune cells induced by LPS sensitively depends on the type of its aggregate structure. In the case of a hexa-acylated bisphosphorylated lipid A structure, high activity is only observed with cubic inverted aggregates. Furthermore, addition of antimicrobial agents (such as polymyxin B) leads to a nearly complete inhibition of cytokine production, whereas the reduction in the Limulus assay is much lower. These data are important since a reliable determination of endotoxin concentrations, in particular with respect to its ability to elicit severe infections, is of high interest.

Influence of composition and preparation parameters on the properties of aqueous monoolein dispersions.

Colloidal cubic phase particles formed in the monoolein/poloxamer/water system are being investigated as potential drug carriers for, e.g., intravenous administration. Preparation methods must, however, still be further developed to reliably yield monoolein dispersions with cubic particles in a size range acceptable for i.v. administration and adequate long-term stability. In this context, the influence of different composition and preparation parameters on the properties of monoolein dispersions prepared by high-pressure homogenization was studied. High pressure homogenization of coarse poloxamer 407-stabilized monoolein/water mixtures leads to dispersions with a large fraction of micrometer-sized particles at low poloxamer concentrations. Higher poloxamer concentrations lead to lower mean particle sizes but the fraction of cubic particles becomes smaller and vesicular particles are observed instead. A study of the characteristics of a dispersion with a standard composition indicated that the homogenization temperature has a much stronger influence on the dispersion properties than the homogenization pressure or the type of homogenizer used. Temperatures around 40-60 degrees C lead to the most favorable dispersion properties. The high temperature sensitivity of the preparation process appears to be at least partly correlated with the phase behavior of the dispersed particles determined by temperature-dependent X-ray diffraction.

Crystallization behavior of supercooled smectic cholesteryl myristate nanoparticles containing phospholipids as stabilizers.

Supercooled smectic nanoparticles based on physiological cholesterol esters are under investigation as a potential novel carrier system for lipophilic drugs. The present study investigates the very complex crystallization behavior of such nanoparticles stabilized with the aid of phospholipids. Phospholipid and phospholipid/bile salt stabilized cholesteryl myristate dispersions were prepared by high-pressure melt homogenization and characterized by particle size measurements, differential scanning calorimetry, X-ray diffraction and electron microscopy. To obtain fractions with very small smectic nanoparticles, selected dispersions were ultracentrifuged. A mixture of cholesteryl myristate and the phospholipid used for the stabilization of the dispersions was also investigated by light microscopy. The nanoparticles usually display a bimodal crystallization event which depends on the thermal treatment and cannot be attributed to crystalline polymorphism. The ratio of the particle fractions crystallizing in the two successive steps strongly depends on the particle size of the dispersions. The presence of larger particles leads to an increased fraction crystallizing at higher temperature and a higher recrystallization tendency upon storage. The observed peculiarities of the crystallization behavior seem to be mainly caused by the presence of particles with different shapes (cylindrical and spherical) as observed in electron microscopy. Alterations in the composition of the nanoparticles may also play a role.

Supercooled smectic nanoparticles: a potential novel carrier system for poorly water soluble drugs.

PURPOSE: The possibility of preparing nanoparticles in the supercooled thermotropic liquid crystalline state from cholesterol esters with saturated acyl chains as well as the incorporation of model drugs into the dispersions was investigated using cholesteryl myristate (CM) as a model cholesterol ester. METHODS: Nanoparticles were prepared by high-pressure melt homogenization or solvent evaporation using phospholipids, phospholipid/ bile salt, or polyvinyl alcohol as emulsifiers. The physicochemical state and phase behavior of the particles was characterized by particle size measurements (photon correlation spectroscopy, laser diffraction with polarization intensity differential scattering), differential scanning calorimetry, X-ray diffraction, and electron and polarizing light microscopy. The viscosity of the isotropic and liquid crystalline phases of CM in the bulk was investigated in dependence on temperature and shear rate by rotational viscometry. RESULTS: CM nanoparticies can be obtained in the smectic phase and retained in this state for at least 12 months when stored at 230C in optimized systems. The recrystallization tendency of CM in the dispersions strongly depends on the stabilizer system and the particle size. Stable drug-loaded smectic nanoparticles were obtained after incorporation of 10% (related to CM) ibuprofen, miconazole, etomidate, and 1% progesterone. CONCLUSIONS: Due to their liquid crystalline state, colloidal smectic nanoparticles offer interesting possibilities as carrier system for lipophilic drugs. CM nanoparticles are suitable model systems for studying the crystallization behavior and investigating the influence of various parameters for the development of smectic nanoparticles resistant against recrystallization upon storage.

Exploring the melting of a semirigid-chain polymer with temperature-resolved small-angle X-ray scattering.

The thermal behavior of semirigid semicrystalline polymers differs significantly from that of flexible-chain polymers. The origin of the differences is believed to lie in the higher energy expenditure associated with the formation of adjacent re-entry folds at the crystalline surface in the case of semirigid chains. The effect of constraints imposed by the interlamellar amorphous regions on the neighboring crystals was studied with temperature-resolved synchrotron radiation small-angle X-ray scattering (SAXS). The analysis of SAXS patterns with a generalized paracrystalline lamellar stack model indicates that melting of a semirigid-chain polymer is not a random process but that the crystals grown in the smallest amorphous gaps melt first. This suggests that the hitherto largely neglected geometrical confinement effects may play an important role in determining the thermodynamic stability of semirigid-chain polymer crystals.

Structural polymorphism and endotoxic activity of synthetic phospholipid-like amphiphiles.

The physicochemical characteristics and in vitro biological activity of various synthetic hexaacyl phospholipid dimers were compared with the respective behavior of bacterial endotoxins (lipopolysaccharide, LPS). The structural variations of the synthetic amphiphiles include different stereochemical (R,S) configurations about their ester- and amide-linkages for the acyl chains and differences in the length of the serine backbone spacer. The temperature of the gel to liquid crystalline phase transition of the acyl chains (T(c)) lies between 10 and 15 degrees C for the compounds with the shortest backbone and decreases rapidly for the compounds with longer backbones. The phase transition enthalpies (8-16 kJ x mol(-1)) are considerably lower than those of lipid A from hexaacyl endotoxins (28-35 kJ x mol(-1)). In contrast, the dependence of T(c) on Mg(2+) and water content shows a behavior typical for endotoxins: a significant increase with increasing Mg(2+) and decreasing water concentrations. The aggregate structure is sensitively dependent not only on the length of the backbone spacer but also on the different stereochemical variations. It can be directly correlated with the biological activity of the compounds. Thus, as with natural lipid A, the capacity to induce cytokine production in mononuclear cells is directly related to the affinity to form nonlamellar cubic or inverted hexagonal H(II) aggregate structures. Together with the data on the transport and intercalation of the dimers into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP), our conformational concept of endotoxicity and cell activation can be applied to these non-LPS structures: endotoxically active compounds incorporate into membranes of immune cells and cause conformational changes at the site of signaling proteins such as Toll-like receptors or K(+)-channels due to their conical molecular shape.

Twists and turns: a tale of two shikimate-pathway enzymes.

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.

Structural characterization of T-protein of the Escherichia coli glycine cleavage system by X-ray small angle scattering.

T-protein, one of the components of the glycine cleavage complex, catalyses the formation of ammonia and methylene-tetrahydrofolate from H-protein-bound intermediate. Native T-protein of the glycine cleavage system from E. coli was efficiently purified using a combination of hydrophobic interaction, gel permeation and ion exchange chromatography. Synchrotron radiation small angle X-ray solution scattering indicates that T-protein has an extended structure in solution. A low resolution model of the protein was constructed ab initio and tentative models of the tertiary structure were built using prediction methods constrained by the scattering data.

Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.

The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.

Solution structure of bacteriophage PRD1 vertex complex.

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity.

The interaction of hemoglobin (Hb) with endotoxins [i.e. with enterobacterial deep rough mutant lipopolysaccharide (LPS) Re and the "endotoxic principle" of LPS, lipid A] was investigated using a variety of physical techniques and with two biological assays, tumor necrosis factor (TNF)-alpha induction in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic binding to the hydrophobic moiety with a slight rigidification of the lipid A acyl chains, and an increase in the inclination of the lipid A backbone with respect to the membrane surface from 35 degrees to more than 40 degrees due to Hb binding, but no change of the predominantly alpha-helical secondary structures of Hb due to LPS binding. From isothermal titration calorimetry, the molar [Hb] : [endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a reorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q224. The LPS-induced TNF-alpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Although a detailed mechanism of action cannot be given, the enhancement of LPS bioactivity can be understood in the light of the previously presented conformational concept; Hb induces an increase in the conical shape of the lipid A moiety of LPS, higher cross-section of the hydrophobic than the hydrophilic part, and of the inclination angle of the diglucosamine backbone with respect to the direction of the acyl chains.

Incorporation of the model drug ubidecarenone into solid lipid nanoparticles.

PURPOSE: The impact of drug incorporation on melt-homogenized tripalmitin nanoparticles is investigated with ubidecarenone as a model drug. The dispersions are studied with respect to their drug loading capacity, localization and physical state of the drug as well as to potential changes of the nanoparticle properties due to interactions between drug and triglyceride matrix. METHODS: The investigations were carried out using photon correlation spectroscopy, differential scanning calorimetry, synchrotron radiation X-ray diffraction, ultracentrifugation, and cryo- and freeze-fracture transmission electron microscopy. RESULTS: Ubidecarenone can be incorporated into the dispersions in concentrations higher than 50% of the dispersed phase. The drug is associated with the nanoparticles such that small drug amounts are bound tightly to the carrier matrix while excess drug adheres as a liquid phase to the crystalline particles. Drug incorporation lowers the crystallization and melting temperature of the particle matrix and accelerates the transition of the triglyceride into the stable beta-polymorph after crystallization. CONCLUSIONS: Drug incorporation may significantly alter important physicochemical parameters of solid lipid nanoparticles. Slow release of ubidecarenone may only be possible for the fraction of drug which is tightly bound to the matrix while the liquid fraction should be rapidly released.

Physico-chemical analysis of lipid A fractions of lipopolysaccharide from Erwinia carotovora in relation to bioactivity.

Highly purified bisphosphoryl, monophosphoryl and dephosphoryl lipids A from Erwinia carotovora with different acylation patterns were characterized physico-chemically. Applying matrix assisted laser desorption/ionization mass spectrometry, the purity of the lipid A fractions was determined, and from monolayer measurements the molecular space requirement was estimated. Fourier transform infrared spectroscopy allowed the elucidation of the gel to liquid crystalline phase transition of the acyl chains as well as the determination of the tilt angle of the diglucosamine backbone with respect to the acyl chain direction applying dichroitic measurements with attenuated total reflectance. With synchrotron radiation small-angle X-ray diffraction the supramolecular aggregate structure was determined, and with fluorescence resonance energy transfer spectroscopy the lipopolysaccharide binding protein induced intercalation of lipid A into a phospholipid matrix corresponding to that of the macrophage membrane was investigated. From the results, a clear dependence of the physico-chemical parameters on the particular lipid A structure can be followed. Furthermore, these parameters correlate well with the biological activities of the various lipids A as deduced from their ability to induce biological activity (Limulus assay and cytokine induction in mononuclear cells). These results contribute to a closer interpretation of the physico-chemical prerequisites for endotoxic activity as found for enterobacterial lipid A.

Conformation of the Drosophila motor protein non-claret disjunctional in solution from X-ray and neutron scattering.

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1.

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei Gö1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.

Determination of domain structure of proteins from X-ray solution scattering.

An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.

The overall conformation of conventional kinesins studied by small angle X-ray and neutron scattering.

The quaternary structures of several monomeric and dimeric kinesin constructs from Homo sapiens and Drosophila melanogaster were analyzed using small angle x-ray and neutron scattering. The experimental scattering curves of these proteins were compared with simulated scattering curves calculated from available crystallographic coordinates. These comparisons indicate that the overall conformations of the solution structures of D. melanogaster and H. sapiens kinesin heavy chain dimers are compatible with the crystal structure of dimeric kinesin from Rattus norvegicus. This suggests that the unusual asymmetric conformation of dimeric kinesin in the microtubule-independent ADP state is likely to be a general feature of the kinesin heavy chain subfamily. An intermediate length Drosophila construct (365 residues) is mostly monomeric at low protein concentration whereas at higher concentrations it is dimeric with a tendency to form higher oligomers.

Twisted plywood pattern of collagen fibrils in teleost scales: an X-ray diffraction investigation.

The distribution and orientation of collagen fibrils, and apatite crystals, in the scales of a bony fish (Leuciscus cephalus) were investigated by X-ray diffraction. The small-angle diffraction patterns obtained with a microfocus scanning setup from most of the examined areas exhibit a distribution of intensity of the collagen reflections according to five preferential orientations, at 36 degrees from one another. It is suggested that the peculiar small-angle X-ray diffraction pattern is due to a plywood arrangement of collagen fibrils in successive layers parallel to the surface of the scale. The fibrils are strictly aligned in each layer and the alignment rotates by 36 degrees in successive layers, according to a discontinuous twist that generates a symmetric plywood pattern. The large spread of the wide-angle reflections does not allow one to distinguish the five directions of orientation in the intensity distribution of the 002 reflection of apatite. However, the patterns recorded from the less ordered regions of the scales display two different orientations of the 002 reflection and allow one to infer a preferential distribution of the apatite crystals with their c-axes parallel to the collagen fibrils. Although much electron microscopic evidence of plywood arrangements in calcified, as well as uncalcified, tissues has been reported, these are the very first diffraction data which unambiguously confirm the presence of these peculiar structures and suggest that this kind of investigation represents a powerful tool with which to study plywood arrangements in biological tissues.