Hydrocarbon chains dominate coupling and phase coexistence in bilayers of natural phosphatidylcholines and sphingomyelins

The structure and thermotropic phase behaviour of aqueous dispersions of egg phosphatidylcholine, egg sphingomyelin, bovine brain sphingomyelin and binary mixtures of phosphatidylcholine and sphingomyelins have been examined by synchrotron X-ray diffraction methods. Small-angle lamellar Bragg peaks and wide-angle X-ray scattering bands have been subjected to peak fitting procedures to identify coexisting gel and fluid as well as fluid-fluid bilayer structures. Molecular species of egg phosphatidylcholine exhibit fluid-fluid immiscibility throughout heating scans from 20 ° to 50 °C. Egg and brain sphingomyelins exhibit gel-fluid bilayer coexistence at temperatures below the main phase transition temperature and fluid-fluid phase coexistence at higher temperatures. Binary mixtures of equimolar proportions of egg phosphatidylcholine and either of the sphingomyelins show gel-fluid phase coexistence at temperatures below the gel phase transition temperature of the respective sphingomyelin. Binary mixtures containing egg sphingomyelin show fluid-fluid immiscibility at all temperatures of the heating scans whereas the fluid phase of mixtures comprising brain sphingomyelin are apparently miscible at all temperatures. An analysis of binary mixtures containing egg sphingomyelin and egg phosphatidylcholine in molar ratios 50:50, 67:33 and 83:17 at 50 °C to identify the composition of the lamellar phases indicated that the two phospholipids are immiscible in bilayers in the fluid phase. The results are discussed in terms of the role of intermolecular hydrogen bonds and hydrocarbon chain composition of sphingomyelins in maintaining coupling across fluid bilayers.     

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1 day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.     

A long ripple phase in DLPC-decylglucoside mixture evidenced by synchrotron SAXS coupled to DSC

For the first time, the secondary ripple phase in a system containing dilauroyl phosphatidylcholine (DLPC) is observed by small-angle X-ray diffraction (SAXS). The SAXS profile exhibits many well-resolved peaks. The fast formation of this phase upon cooling from the liquid crystalline lamellar phase L(alpha) is induced by addition of C10G with molar ratio 0.17< or = R = [C10G]/[DLPC]< or = 0.49. For R < 0.17, the primary P(beta') ripple phase is observed. In contrast to the P(beta') phase, which shows a sawtooth shape, the secondary ripple structure is thought to be symmetric. The ripple length (190 angstroms) and the bilayer spacing (74 angstroms) are larger than in the primary ripple phase. Lattice parameters of the new long ripple phase, which are quite insensitive to temperature, vary slightly linearly with R. In this study, structural and thermodynamic changes within the samples were followed as a function of temperature by time-resolved X-ray diffraction coupled to DSC.     

Small-angle X-ray scattering studies of giant haemoglobin from the annelid Tylorrhynchus heterochaetus

The extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus was studied in solution by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 10.8±0.2 nm and a maximum intraparticular distance of 29.5±0.5 nm. Models which fit well the experimental data and reflect also the biochemical structure especially the known number of polypeptide chains are presented.     

Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex

Clostridium botulinum produces the botulinum neurotoxin (BoNT). Previously, we provided evidence for the “building-block” model of botulinum toxin complex (TC). In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA), yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three “arms” of the HA-33/HA-17 trimer (two HA-33 and one HA-17) further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS). The mature three-arm L-TC exhibited the shape of a “bird spreading its wings”, in contrast to the model having three “arms”, as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.     

1964: The first model for the shape of a transfer RNA molecule. An account of an unpublished small-angle X-ray scattering study

The shape of non-fractionated Escherichia coli transfer RNA molecules in solution was investigated using small-angle X-ray scattering during the years 1960-1962 at the Centre de Recherche sur les Macromolécules in Strasbourg. The innermost region of the scattering curve yielded the average molecular weight (Mr) and the radius of gyration (Rg) of the particles, whereas the experimental data at large angles could be approximated at best by the scattering curve of a kinked rod-shaped molecule. The simplest model that was compatible with Mr, Rg, and the mass per unit length of the rod was a boomerang-shaped particle made of two double helical stems connected by a sharp kink. This model that eventually proved similar to the high-resolution L-shaped structure, was presented in my Ph.D. dissertation (J. Witz, Etude de la structure de quelques polynucléotides en solution par diffusion centrale des rayons X, Ph.D. dissertation, University of Strasbourg, France, 1964) but has never been published in detail. It is the purpose of this note to recall this story.     

Fluorescence temperature jump relaxations of dansylphosphatidylethanolamine in aqueous dispersions of dipalmitoylphosphatidylcholine during the gel to liquid-crystal transition

The change of fluorescence of the probe dansylphosphatidylethanolamine embedded in multilamellar liposomes of dipalmitoylphosphatidylcholine was used to investigate the dynamics of the thermotropic gel to liquid-crystalline phase transition by use of the temperature-jump technique. The results are discussed and compared to published observations on the same system in which the phenomenon was reported by turbidity changes.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 °C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH₂, CH, and terminal CH₃ groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 degrees C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH2, CH, and terminal CH3 groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Effect of the carbohydrate side-chain on the conformation of a glycoconjugate polystyrene in aqueous solution

An oligomaltose-carrying polystyrene “glycoconjugate polystyrene” was synthesized by the homopolymerization of 4-vinylbenzylamine oligomaltonic amides, derived from maltose, maltotriose, maltopentaose, and maltoheptaose. The resultant amphiphilic glycoconjugate polystyrenes were dissolved in 0.1 M aqueous urea, and their structures characterized by small-angle X-ray scattering and molecular modeling. “Glycoconjugate polystyrene” was found to behave as a “molecular bottle brush”, composed of a large pseudo-helical polystyrene backbone and carbohydrate brushes. A large pseudo-helical polystyrene backbone is formed by a random sequence of TT, TG, and/or TTGG. The results indicate that the cross-section of a backbone chain with smaller oligosaccharide side-chains is obliged to expand more than that with longer side-chains. Even with rigid hydrophilic pendant oligosaccharide chains, the larger pseudo-helix of the main chain could orient the side-chains so as to envelop the hydrophobic backbone in aqueous solution. Thus the conformation of the main chain is determined not only by the chemical nature of an oligosaccharide chain but also by its length.     

SANS/SAXS study of the BSA solvation properties in aqueous urea solutions via a global fit approach

We report on the solvation properties and intermolecular interactions of a model protein (bovine serum albumine, BSA) in urea aqueous solutions, as obtained by combining small-angle neutron and X-ray scattering experiments. According to a global fit strategy, all the whole set of scattering curves are analysed by considering a unique model which includes the BSA structure, the protein-protein interactions and the thermodynamic exchange process of water/urea molecules at the protein solvent interface. As a main result, the equilibrium constant that accounts for the difference in composition between the bulk solvent and the protein solvation layer is derived. Results confirm that urea preferentially sticks to the protein surface, inducing a noticeable change in both the repulsive and the attractive interaction potentials.     

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila.Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent.To obtain information about the IDR regions, we studied full-length SuFu’s structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.     

On the stability of the ripple phase in the DPPC/PLPC/water ternary system

The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the P_β′ ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % ²H₂O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.     

Structure of the stable and metastable ripple phase of dipalmitoylphosphatidylcholine

Dipalmitoylphosphatidylcholine (DPPC) dispersed in excess water forms a stable ripple phase upon heating from the gel phase and a metastable ripple phase P (mst) upon cooling from the liquid crystalline phase. The X-ray diffraction pattern of P (mst) displays several reflections in the range from 1/25 to 1/2.8 nm^–1, which can all be indexed on a two-dimensional monoclinic lattice (space group p2) with a=26.2, b=8.63 nm and =107°. In contrast to the stable ripple phase, which shows a sawtooth like surface profile and an almost constant bilayer thickness, the electron density map of the metastable ripple phase shows an almost symmetric surface profile with a modulation length of 26.2 nm. The lipid bilayer thickness varies from 3.9 to 4.4 nm, which most likely arises from a continuous periodic change of the tilt of the chains to the surface normal of between 30 and 40 degrees. A further important feature of the structure is the staggered stacking of the bilayers with water pockets enclosed.     

Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate

Apomyoglobin undergoes a two-step unfolding transition when the pH is lowered from 6 to 2. The partly folded intermediate (1) state at pH 4 and low ionic strength has properties of a molten globule. We have studied structural features of this state, its compactness, content of secondary structure, and specific packing of aromatic side chains, using dynamic light scattering, and small-angle X-ray scattering and far- and near-ultraviolet circular dichroism spectroscopy. Particular attention was paid to temperature-dependent structural changes. The results are discussed with reference to the native-like (N) state and the highly unfolded (U) state. It turned out that the I-state is most compact near 30°C, having a Stokes radius 20% larger and a radius of gyration 30% larger than those of the N-state. Both cooling and heating relative to 30°C led to an expansion of the molecule, but the structural changes at low and high temperatures were of a different kind. At temperatures above 40°C non co-operative melting of structural elements was observed, while the secondary structure was essentially retained on cooling. The results are discussed in context with theoretical predictions of the compactness and the stability of apomyoglobin by Alonso et al. [Alonso, D. O. V., Dill, K, A., and Stigler, D. (1991) Biopolymers 31:1631–1649]. Comparing the I-state of apomyoglobin with the molten globules of -lactalbumin and cytochrome c, we found that the compactness of the molten globule states of the three proteins decreases in the order -lactalbumin > apocytochrome c > apomyoglobin. While -lactalbumin and cytochrome c are rather homogeneously expanded, apomyoglobin exhibits a non uniform expansion, since two structural domains could clearly be detected by small-angle X-ray scattering.Abbreviations CD circular dichroism - DLS dynamic light scattering - SAXS small-angle X-ray scattering - N, 1, and U the native, intermediate, and unfolded forms of apomyoglobin Correspondence to: G. Damaschun     

A calorimetric and fluorescent probe study of the gel-liquid crystalline phase transition in small, single-lamellar dipalmitoylphosphatidylcholine vesicles.

The results of a calorimetric and fluorescent probe study of the thermotropic behavior of various types of dispersions of dipalmitolphosphatidylcholine bilayer vesicles are reported. Bangham-type, multilamellar vesicles exhibit tow distinct phase transitions at 34.6 and 41.2 degrees C. On the other hand, single-lamellar spherical vesicles appear to exhibit a single transition at 37 degrees C. The single-lamellar vesicles are thermodynamically unstable below 27 degrees C and slowly transform into a multilamellar structure with a single phase transition of 41.2 degrees C. These transformed structures resemble, but are not identical with, Bangham-type vesicles. An experimentally testable thermodynamic and kinetic model based upon these results is developed.     

The effect of cholesterol in small amounts on lipid-bilayer softness in the region of the main phase transition

The temperature dependence of the small-angle neutron scattering from aqueous multilammellar DMPC lipid bilayers, containing small amounts of cholesterol, is analyzed near the main phase transition by means of a simple geometric model which yields the lamellar repeat distance, the hydrophobic thickness of the bilayer, the interlamellar aqueous spacing, as well as fluctuation parameters. The observation of anomalous swelling behavior in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Our results indicate that the effect of small amounts of cholesterol, ≲3 mole%, is a softening of the bilayers in the transition region, whereas cholesterol contents above this range lead to the well-known effect of rigidification. The possible biological relevance of this result is discussed. Received: 24 October 1996 / Accepted: 9 December 1996     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

The evidence of large-scale DNA-induced compaction in the mycobacterial chromosomal ParB

The bacterial chromosome trafficking apparatus or the segrosome participates in the mitotic-like segregation of the chromosomes prior to cell division in several bacteria. ParB, which is the parS DNA-binding component of the segrosome, polymerizes on the parS-adjacent chromosome to form a nucleoprotein filament of unknown nature for the segregation function. We combined static light scattering, circular dichroism and small-angle X-ray scattering to present evidence that the apo form of the mycobacterial ParB forms an elongated dimer with intrinsically disordered regions as well as folded domains in solution. A comparison of the solution scattering of the apo and the parS-bound ParBs indicates a rather drastic compaction of the protein upon DNA binding. We propose that this binding-induced conformational transition is priming the ParB for polymerization on the DNA template.     

Complex formation of potassium salt of highly fatty acid with hemagglutinin protein in influenza virus via exothermic interaction

In our previous study, we found highly fatty acid salts, which are a skin-friendly soaps, had a high ability to inactivate the influenza virus. In order to elucidate the mechanism of inactivation of influenza virus, we investigated interactions and complex formation of potassium tetradecanoate (C14K) as a highly fatty acid salt with a virus particle (VP) derived from avian influenza virus by using isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). ITC showed C14K attractively interacted with hemagglutinin protein (HA) which exists in the envelop of VP. SAXS analyses revealed C14K formed highly ordered complex with HA through the attractive interaction. Since the HA is responsible for cell entry events, inactivation of influenza viruses by highly fatty acid salts are derived owing to HA inhibition of influenza viruses through the complex formation. Time-resolved SAXS measurements elucidated the complex formation was completed within 40 s after mixing aqueous solutions of C14K and VP. This result strongly suggests that hand-washing with a highly fatty acid salts is an effective measure to prevent infection with influenza virus without causing rough hands.