The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction,circular dichroism spectrum and absorption spectrum studies

The phase transition of the purple membrane observed by differential scanning calorimetry (Jackson, M.B. and Sturtevant, J.M. (1978) Biochemistry 17, 911–915) has been investigated by X-ray diffraction, circular dichroism and absorption spectrum, in comparison with the phase transition in the brown holo-membrane. The two-dimensional crystal of bacteriorhodopsin transformed into two-dimensional liquid around 74–78°C in the purple membrane and around 50–60°C in the brown holo-membrane. The X-ray diffraction patterns obtained at 78°C for the purple membrane and at 60°C for the brown holo-membrane exhibit several broad peaks. Analysis of the pattern suggests that bacteriorhodopsin molecules aggregate in trimers even above the phase transition temperature. The negative circular dichroism band in the visible region is still present at 80°C in the purple membrane and at 60°C in the brown holo-membrane, but becomes negligibly small at 70°C in the brown holo-membrane. The 560 nm absorption peak due to bacteriorhodopsin changes its position and height drastically around 80°C in the brown holo-membrane as in the purple membrane. X-ray diffraction studies have been made on membranes of total lipids extracted from the purple membrane. No indication of the phase transition has been found between ?81°C and 77°C.     

The crystal structure and physicochemical characteristics of 2-hydroxy-N-[3(5)-pyrazolyl]-1,4-naphthoquinone-4-imine,a new antitrypanosomal compound

This study was designed to investigate the physical characteristics and crystalline structure of 2-hydroxy-N-[3(5)-pyrazolyl]-1,4-naphthoquinone-4-imine (PNQ), a new active compound against Trypanosoma cruzi, the causative agent of American trypanosomiasis. Methods used included differential scanning calorimetry, thermogravimetry, hot stage microscopy, polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and high-resolution X-ray powder diffraction (HR-XRPD). According to PLM and HR-XRPD data, PNQ crystallized as red oolitic crystals (absolute methanol) or prisms (dimethyl sulfoxide [DMSO]-water) with the same internal structure. The findings obtained with HR-XRPD data (applying molecular location methods) showed a monoclinic unit cell [a = 18.4437(1) A, b = 3.9968(2) A, c = 14.5304(1) A, alpha = 90 degrees , beta = 102.71(6) degrees , gamma = 90 degrees , V = 1044.9(1) A(3), Z = 4, space group P2(1)/c], and a crystal structure (excluding H-positions) described by parallel layers in the direction of the b-axis, with molecules held by homochemical (phenyl-phenyl and pyrazole-pyrazole) van der Waals interactions. In addition, FTIR spectra displayed the NH-pyrazole stretch overlapped with the OH absorption at 3222 cm(-1), typical of -NH and -OH groups associated through H-bondings; and a carbonyl stretching absorption at 1694 cm(-1), indicating a nonextensively H-bonded quinonic C=O, which was in accordance with the solved crystal structure of PNQ. The existence of such cohesive forces shed light on the thermoanalytical data, which revealed that PNQ is a stable solid, unaffected by oxygen that decomposed without melting above 260 degrees C.     

Coexistence of two domains in intercellular lipid matrix of stratum corneum

The outermost layer of the skin, the stratum corneum (SC), is composed of corneocytes and an intercellular lipid matrix. The matrix acts as both the main barrier and also as the pathway of water, drugs, etc. across the SC. In the mammalian SC, the longitudinal arrangement of the lipid molecules, consisting of long and short lamellar structures with repeat distances of about 13 nm and 6 nm, respectively, has been observed by small-angle X-ray diffraction. In the lateral arrangement of the lipid molecules, hexagonal and orthorhombic hydrocarbon-chain packing has been observed by wide-angle X-ray diffraction. From the systematic study of the temperature dependence of simultaneous small- and wide-angle X-ray diffraction patterns, we demonstrate that the intercellular lipid matrix forms two domains, which consist at room temperature of a long lamellar structure with hexagonal hydrocarbon-chain packing and a short lamellar structure with orthorhombic hydrocarbon-chain packing.     

Influence of precipitating agents on thermodynamic parameters of protein crystallization solutions

X‐ray crystallography is the most powerful method for determining three‐dimensional structures of proteins to (near‐)atomic resolution, but protein crystallization is a poorly explained and often intractable phenomenon. Differential Scanning Calorimetry was used to measure the thermodynamic parameters (ΔG, ΔH, ΔS) of temperature‐driven unfolding of two globular proteins, lysozyme, and ribonuclease A, in various salt solutions. The mixtures were categorized into those that were conducive to crystallization of the protein and those that were not. It was found that even fairly low salt concentrations had very large effects on thermodynamic parameters. High concentrations of salts conducive to crystallization stabilized the native folded forms of proteins, whereas high concentrations of salts that did not crystallize them tended to destabilize them. Considering the ΔH and TΔS contributions to the ΔG of unfolding separately, high concentrations of crystallizing salts were found to enthalpically stabilize and entropically destabilize the protein, and vice‐versa for the noncrystallizing salts. These observations suggest an explanation, in terms of protein stability and entropy of hydration, of why some salts are good crystallization agents for a given protein and others are not. This in turn provides theoretical insight into the process of protein crystallization, suggesting ways of predicting and controlling it. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 642–652, 2016.     

An estimate of the minimum amount of fluid lipid required for the growth of escherichia coli

The lipid phase transition of Escherichia coli was studied by high sensitivity differential scanning calorimetry. A temperature sensitive unsaturated fatty acid auxotroph was used to obtain lipids with subnormal unsaturated fatty acid contents. From these studies it was concluded that E. coli can grow normally with as much as 20% of its membrane lipids in the ordered state but that if more than 55% of the lipids are ordered, growth ceases. Studies with wild-type cells show that the phase transition ends more than 10°C below the growth temperature when the growth temperature when the growth temperature is either 25°C or 37°C.     

Interaction of dipalmitoyl phosphatidylserine with ethanol: induction of an ordered gel phase at room temperature

Using differential scanning calorimetry and small and wide-angle X-ray diffraction, we show that, unlike the saturated phosphatidylcholines, for which ethanol induces chain interdigitation in the gel state, and unlike natural phosphatidylserine in which the gel state is almost unaffected by the addition of ethanol, dipalmitoyl phosphatidylserine (DPPS) assumes an ordered structure after incubation at room temperature in the presence of as little as 5% (v/v) ethanol. In the liquid crystalline state, a progressive decrease in the interbilayer spacing is observed as a function of ethanol concentration, similar to what is found for natural phosphatidylserine (PS) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS). The 0.37 molar fraction of cholesterol in the DPPS dispersion in the presence of 10% (v/v) ethanol, does not prevent the formation of the ordered gel.     

Physicochemical characterization of silicon-containing glycolipids by DSC,FT-Raman spectroscopy and X-ray diffraction

Derivatives of dimethylalkylchlorosilanes are novel substances which may be used in formulations for drug targeting. In order to design their properties it is essential to perform physicochemical characterization. For this purpose, a combination of differential scanning calorimetry (DSC), FT-Raman spectroscopy and X-ray diffraction is well suited. For the starting material dimethyloctadecylchlorosilane (DMOC), the assignment of Raman bands is discussed. The influence of sugar-containing head groups on the structures of the hydrocarbon chains of 1-O-(dimethyldodecylsilyl)-[2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside] and 1-O-(dimethyloctadecylsilyl)-[2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside] was investigated using the band position of the symmetric methylene mode. The temperature dependence of conformationally sensitive bands in the CH(2)-stretching region (2800-2900 cm(-1)), C-C-stretching region (1000-1150 cm(-1)) and CH(3)-rocking region (830-900 cm(-1)) was studied to characterize the state of order of the alkyl chains. Using X-ray diffraction, the repeating distances of layered structures was determined. The phase transitions occurring were found to be completely reversible. The subcell of DMOC shows an orthorhombic perpendicular packing structure in the crystalline state.     

Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH 7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22 μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

A microcalorimetric study of collagen hydration in the temperature range 20–100°C by measuring the enthalpy of water evaporation from sample

A new definition of the term “biopolymer hydration,” based on thermodynamic characteristics of water evaporation from biological preparations, is proposed. A new method for investigation of bound water, based on precise measurement of the enthalpy of water evaporation from the sample, using differential scanning micro-calorimetry, is developed. Adequacy of the new approach for estimating the water state in biopolymers and at various levels of structural organization of biosystems is demonstrated by study of collagen fibers as an example.     

Polymorphism of ceramide 3. Part 1: an investigation focused on the head group of N-octadecanoylphytosphingosine

The thermotropic phase behaviour of the ceramide N-octadecanoylphytosphingosine (CER3) was investigated using differential scanning calorimetry, X-ray powder diffraction and FT-IR spectroscopy. CER3 was shown to be a polymorphic substance depending on the crystallisation conditions. Three different solid states were found. The FT-IR results elucidate changes in the hydrogen bonding interactions of the ceramide head group. It was shown that the amide I and the amide II vibration bands are quite sensitive to the phase transitions of CER. There are clear shifts in the band positions of those bands passing the phase transitions. Furthermore, changes were observed in the NH- and OH- stretching region. The study shows that there are strong inter- and intramolecular hydrogen bonds between hydroxy groups in the ceramide head group. There are also strong hydrogen bonds to the amide oxygen as shown by the band positions of the amide vibrations. The H-bonding network and conformation of the head group of CER3 alters due to the phase transitions.     

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.     

Crystal structure of amylose complexes with small ligands

Amylose complexes were prepared, as lamellar single crystals and polycrystalline powders, from aqueous solutions by adding small flavor molecules. The morphology, crystal structure, and thermal properties of complexes with fenchone, menthone, and geraniol were determined using transmission electron microscopy, wide-angle X-ray diffraction, and differential scanning calorimetry. The crystal structure was found to be similar to that of V amylose complexes with isopropanol. This implies that the crystallosolvates contain sixfold helices packed in orthorhombic unit cells, with the ligands possibly lying in the interhelical space. Different drying procedures were also studied leading to less resolved X-ray diagrams. The thermoanalysis confirmed that complexes with a relatively high crystallinity were formed.     

Triglycerides obtained by dry fractionation of milk fat: 2. Thermal properties and polymorphic evolutions on heating

The crystallographic and thermal properties of milk fat and fractions were investigated on heating using the coupling of synchrotron X-ray diffraction with differential scanning calorimetry. We showed that re-crystallisations occurred during the heating of the stearin and the olein fractions with the formation of a β′ 2L (41.1-42.6 Å) structure and a β′ 3L (66 Å) structure, respectively. By creating a quantified solid-liquid phase behaviour versus temperature diagram, the amount of the solid and liquid phases and the relative proportion of each of the crystalline structures within the solid phase were determined.     

Effect of urea, dimethylurea, and tetramethylurea on the phase behavior of dioleoylphosphatidylethanolamine

The phase behavior of dioleoylphosphatidylethanolamine in aqueous solutions of urea, N,N'-dimethylurea (DMU), and N,N,N',N'-tetramethylurea (TMU) has been characterized by synchrotron X-ray diffraction and differential scanning calorimetry. All three solutes stabilize the lamellar liquid-crystalline phase at the expense of lamellar-gel phase and inverted hexagonal phase of the phospholipid when present in concentrations up to 3 M. X-ray diffraction data demonstrated that the repeat spacing of DOPE increased with increasing urea concentration, but decreased as the DMU and TMU concentrations increased. The repeat spacing of DOPE in the liquid-crystal phase dispersed in the three solutes is d(urea)>d(DMU)>d(TMU). The molecular mechanisms underlying these observations are discussed in terms of either membrane Hofmeister effect, where urea acts as a water structure breaker, or a direct insertion effect of the amphiphilic DMU and TMU molecules into the lipid head groups in the interfacial region of the phospholipid bilayer.     

Laser light scattering of high amylose and high amylopectin materials in aqueous solution, effect of storage time

Light scattering techniques were used for structural characterization of starches with diverse amylose and amylopectin level, dissolved in water by microwave heating in a high pressure vessel and stored during different times. In general, apparent molar mass ( ), gyration radius ( ) and hydrodynamic radius ( ) values decreased when storage time increased. This could be due to depolymerization of the samples during the storage time. The fractal dimension obtained from the – relationship showed that the samples presented, in general, a globular structure, with a higher level of branching when amylopectin level in the sample increased. The particle scattering factors and Kratky plots, well suited for studying the internal structure of a macromolecule, showed a depolymerization when storage time increased. The ν_RH values for Eurylon 5 (0.56) and Eurylon 7 (0.58) starches were close to the values reported for linear chains. For amylopectin (0.09) and normal corn starch (0.10) the ν_RH values were lower; these values would define a highly branched structure. The relaxation rate distribution of the samples showed that there are changes in the internal structure when storage time increases, and that these changes depend on amylose and amylopectin level present in the sample. The ρ values for the samples analyzed were between 0.88 and 1.3; these values are characteristic of a sphere or globular structure.     

Milk fat and primary fractions obtained by dry fractionation 1. Chemical composition and crystallisation properties

The chemical composition and crystallisation properties of milk fat and its primary fractions, obtained by dry fractionation at 21 degrees C, were investigated. The solid fraction (stearin) and the liquid fraction (olein) displayed a different triacylglycerol (TG) composition. Stearin fraction was enriched in long-chain fatty acids, whereas olein fraction was enriched in short-chain and unsaturated fatty acids. Crystallisation properties of milk fat, and both the stearin and olein fractions were studied on cooling at |dT/dt|=1 degrees C min(-1) by differential scanning calorimetry and time-resolved synchrotron X-ray diffraction (XRD) at small and wide angles. Two main types of crystals corresponding to double chain length structures were characterised in the stearin fraction: alpha 2L(1) (47.5 Angstrom) and beta' 2L(2) (41.7 Angstrom). A triple chain length structure was formed in the olein fraction: alpha 3L (72.1 Angstrom). Crystallization of milk fat showed the formation of two 2L (47.3 and 41.6 Angstrom) and one 3L (72.1 Angstrom) lamellar structures with an hexagonal packing (alpha form). A schematic representation of the 3L packing of olein fraction was proposed to explain how a wide diversity of TG can accommodate to form a lamellar structure with a thickness of 72 Angstrom. Furthermore, the sharpness of the small-angle XRD lines associated to the alpha form was explained by the formation of liquid crystals of smectic type.     

Changes in the physicochemical characteristics of rabbit sclera upon scleral reinforcement

Biochemical analysis and differential scanning calorimetry demonstrated that the connective tissue (capsule) formed around a reinforcing scleroplastic implant is similar to intact sclera, its main component being type I collagen organized in perfect fibrils with cross-linking sufficient for normal thermomechanical properties. DSC also revealed a fraction of collagen with heat-labile ‘immature’ cross-links around implants containing a stimulatory plant product Panaxel, which suggested high synthetic activity of fibroblasts.