Thermally reversible hydration of beta-chitin

Thermally induced transition between anhydrous and hydrated forms of highly crystalline beta-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet beta-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 degrees C on heating and one broad exothermic peak at between 60 and 0 degrees C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet beta-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the beta-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process.     

The structure of D-erythro-C18 ceramide at the air-water interface

X-ray reflectivity (XR) and diffraction at grazing angles of incidence (GID) were conducted to determine the structure of synthetic D-erythro C18-ceramide films at the air-water interface at various surface pressures (pi). Analysis of the GID reveals that the monomolecular film, at the crystalline phase (pi > 0 mN/m), is predominantly hexagonal. In this crystalline phase, the analysis of the reflectivity yields an electron density profile that consists of three distinct homogeneous slabs, one associated with the headgroup region and the other two with the hydrocarbon chains. At large molecular areas (pi approximately 0), isolated crystalline domains coexist with two-dimensional gas phase. Within the crystalline domains, we find an orthorhombic arrangement of the chains that coexists with the hexagonal symmetry. It is argued that the two-dimensional orthorhombic crystals are induced by hydrogen bonding between headgroups even at very low surface pressures. Although their structure is incommensurate with the simple hexagonal arrangement, they act as nucleation centers for the conventional hexagonal phase which dominates at high pi.     

Native fat globules of different sizes selected from raw milk: thermal and structural behavior

The aim of this study was to characterize differences in the thermal and structural behavior between different sized native milk fat globules. A novel microfiltration process permits the selection of native small fat globules (SFG, 1-3 microm) and large fat globules (LFG, >5 microm) in raw milk, that were analyzed by X-ray diffraction (XRD) coupled to differential scanning calorimetry (DSC). There were no major differences in triglyceride crystalline structures between SFG and LFG, after eliminating thermal history and the influence of cooling rates. The three main 3L and 2L crystalline structures appearing under slow cooling existed regardless of globule size. The supercooling increased for the SFG, mainly due to heterogeneous nucleation in winter milk, and also to compositional variations in spring milk. Differences appeared regarding stabilized crystalline forms at 20 degrees C and subsequent cooling: the SFG contained less 2L triglyceride structures than the LFG. These results can be important in dairy manufactures using tempering periods.     

Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection.

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na+ + K+)-stimulated Mg2-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61 degrees C, about 20 degrees C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28 degrees C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

Preliminary Studies on the Denaturation of Cross-Linked Lysozyme Crystals

When monoclinic lysozyme crystals are fully cross-linked with glutaraldehyde, and then the protein molecules are denatured while in the crystalline state, a single crystal-gel is formed which is a jelly-like crystal of a denatured protein molecule. It is highly disordered, but has crystalline optical and morphological properties and can be renatured to a cross-linked crystal resembling the original crystal as determined from the X-ray diffraction pattern. Experiments with the following denaturants are described: guanidinium chloride, bromoethanol, urea, and lithium chloride.     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular conformation prostaglandin A1 monoclinic crystalline polymorph.

The molecular conformation of the monoclinic crystalline polymorph of prostaglandin A1 has been determined by X-ray diffraction techniques. The space group is P21 with a = 13.637(2), b = 7.567(1), c = 10.576(2) A, beta = 107.37(3) degrees; Dc = 1.073 g.cm-3 for Z = 2. The molecular conformation is characterized by the nearly parallel arrangement of the C1-C7 and C13-C20 side chains, with a general flattening of the overall structure when compared with the orthorhombic polymorph. The cyclopentenone moiety assumes a C8 envelope conformation with C8 and O9 displaced +0.29 A and -0.18 A from the C9-C10=C11-C12 plane respectively. Concerted, small varations of the torsion angles, primarily about the C8-C12, C14-C15 and C16-C17 bonds, bring the monoclinic and orthorhombic conformations into coincidence.     

Temperature-induced vertical shift of proteins in membranes

Thermotropic changes in the transverse order of microsomal membranes isolated from Tetrahymena are revealed by low-angle X-ray diffraction. These are correlated with the lateral order of the membrane lipids by wide-angle X-ray diffraction. Upon lowering the temperature from 28 °C to 2 °C, the Bragg period of the membrane stack reveals an abrupt increase of ~3.0 nm at ~19 °C, which is reversible upon reheating to 28 °C. This is coupled with an alteration in the electron density profile, revealing a shift of mass from the hydrophobic core towards one of the two hydrophilic surfaces. Between 35 °C and 0 °C, the membrane lipids undergo a broad, thermotropic “two-stage” liquid crystalline ? crystalline phase separation with a “breakpoint” at ~18 °C. This breakpoint signals an abrupt lipid redistribution, presumably due to a change in the composition of the two coexisting liquid crystalline and crystalline lipid phases. We conclude: (1) the temperature-induced mass shift reflects a shift in the transverse partition of proteins in membranes; (2) this is triggered by an abrupt lipid redistribution occurring during a broad liquid crystalline ? crystalline phase separation.     

A time-resolved synchrotron X-ray study of a crystalline phase bilayer transition and packing in a saturated monogalactosyldiacylglycerol-water system

The structural changes associated with a phase transition between the gel-phase bilayer (L_β) in which the acyl chains pack in a hexagonal subcell, and a crystalline bilayer phase (L_C1) where the acyl chains are packed in an orthorhombic subcell in a saturated monogalactosyldiacylglycero-water system are reported. The phase change is cooperative and takes place isothermally after the lamellar-gel phase has been held at 20°C for about 8 min. The transformation of the acyl chain subcell from hexagonal to orthorhombic induces a change in diffraction maxima observed in the region 0.6–0.7 nm which is interpreted as a change in packing of the galactose residues from an orthorhombic to hexagonal subcell. We conclude that the rearrangement of the acyl chains into a more closely packed subcell requires the head groups to reorient to reduce the steric hindrance between the bulky galactose residues.     

Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.     

Mechanism and kinetics of the subtransition in hydrated L-dipalmitoylphosphatidylcholine

The mechanism of the subtransitions (Lc to L beta') in L-dipalmitoylphosphatidylcholine bilayers in excess water has been investigated by time-resolved X-ray diffraction using synchrotron radiation. The temperature dependence of the diffraction patterns closely correlate with the asymmetric excess specific heat variation recorded by differential scanning calorimetry. During the subtransition two prominent wide-angle reflections, characteristic of the low-temperature crystalline phase, Lc, gradually change such that a sharp peak at a spacing of 0.430 nm decreases in intensity and ultimately disappears while a broader peak initially located at 0.375 nm progressively shifts to an eventual spacing of 0.410 nm. This behaviour is interpreted as a lateral deformation of the acyl chain packing subcell as the chains begin to rotate until a state is reached where the chains pack on a regular hexagonal array characteristic of the L beta phase. An increase in lamellar repeat distance from 6.0 to 6.4 nm takes place simultaneously with the acyl chain rearrangement at relatively low (5 K/min) as well as high (6 K/s) heating rates. As judged from the shape of the wide-angle peak, transformation to L beta' phase occurs some minutes after transition to the L beta phase. The X-ray data characterise the subtransition as a continuous (second order) phase transition in which a presumably orthorhombic subcell is transformed into a hexagonal subcell in a gradual process. In temperature jump experiments at 6 K/s between 0 degree C and 80 degrees C the relaxation time of the subtransition was found to be about 5 s while the relaxation time of the main gel to liquid-crystalline transition was about 2 s.     

The existence of a highly ordered phase in fully hydrated dilauroylphosphatidylethanolamine

Dilauroylphosphatidylethanolamine dispersion forms a crystalline phase at physiological pH and temperature and in the presence of excess water. This phenomenon was observed and studied by differential scanning calorimetry, scanning densitometry and X-ray diffraction. The crystalline phase is stable at pH 5.5-9.5 and below 40 degrees C. The crystalline phase formed at pH 5.5 and pH 9.5 index according to orthorhombic cells with a = 9.41, b = 8.15, c = 46.0 and a = 9.33, b = 8.05, c = 45.8 (A), respectively. Around 43 degrees C, the crystalline phase is transformed into a multilayer liquid crystal phase. Cooling from 44 degrees C results in the disappearance of the original transition at 43 degrees C and the appearance of a second transition at around 30 degrees C. Below 30 degrees C the lipid forms a gel phase. This gel phase is metastable at pH 5.5 and a crystalline phase may be recovered from it by dispersing or aging methods. Suspensions of dilauroylphosphatidylethanolamine show similar phase transition behaviour at pH 5.5 and pH 9.5, although the transitions are somewhat broader at the higher pH. The thermotropic phase behaviour of dilauroylphosphatidylethanolamine dispersions may be governed by changes in hydration.     

X-ray absorption spectroscopy as a probe of microbial sulfur biochemistry: the nature of bacterial sulfur globules revisited

The chemical nature of the sulfur in bacterial sulfur globules has been the subject of controversy for a number of years. Sulfur K-edge X-ray absorption spectroscopy (XAS) is a powerful technique for probing the chemical forms of sulfur in situ, but two groups have used it with very different conclusions. The root of the controversy lies with the different detection strategies used by the two groups, which result in very different spectra. This paper seeks to resolve the controversy. We experimentally demonstrate that the use of transmittance detection for sulfur K-edge XAS measurements is highly prone to spectroscopic distortions and that much of the published work on sulfur bacteria is very likely based on distorted data. We also demonstrate that all three detection methods used for X-ray absorption experiments yield essentially identical spectra when the measurements are carried out under conditions where no experimental distortions are expected. Finally, we turn to the original question—the chemical nature of bacterial sulfur. We examine isolated sulfur globules of Allochromatium vinosum and intact cells of a strain of magnetotactic coccus and show that XAS indicates the presence of a chemical form of sulfur resembling S₈.     

Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na⁺ + K⁺)-stimulated Mg²⁺-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61°C, about 20°C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28°C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

A new approach to the remediation of heavy metal liquid wastes via off-gases produced by Klebsiella pneumoniae M426

When the off-gas from an aerobic culture of Klebsiella pneumoniae M426 grown in the absence of added heavy metals was passed through a solution of Hg(2+), Cd(2+), Pb(2+), or Cu(2+) a yellow-white (Hg), white (Cd, Pb), or blue (Cu) precipitate was formed. Metal removal from solution was >97% within 2 h at initial concentrations of (as metal): Hg, 8.5; Cd, 12.6; Pb, 7.8; Cu, 9.5 mg/mL. Mercury was removed from solution at pH 2 and in up to 1 M NaCl. Energy dispersive X-ray microanalysis (EDX) of the precipitates showed them to comprise metal, sulfur and carbon in the case of Hg, Cd, and Pb, and, in the case of Cd and Pb, also oxygen. The pH of the solution increased by 1-2 units at an initial pH of 7 and by 4-5 units at an initial pH of 2. In the case of cadmium and lead, the presence of crystalline metal carbonates and hydroxides was confirmed by X-ray powder diffraction (XRD) analysis and additional peaks were seen which could not be assigned to known compounds in the diffraction file database. In the case of copper, hydroxides, and a form of copper sulfate, were found. In the case of mercury the XRD patterns could not be assigned to any known compound, except for HgCl in the solution at the acidic initial pH. The absence of sharp peaks in the pattern for the Hg-precipitate was indicative of poorly crystalline, nanocrystalline or amorphous material. The unknown mercury compound, since it contained non-carbonate carbon, was suggested to be derived from a volatile organothiol in the gases evolved from the culture. Analysis of the culture head gas by GC-MS showed the presence of dimethyldisulfide as a likely precipitant. No sulfur compound was found using XRD analysis in the case of cadmium and lead, although EDX analysis suggested this as a major component and the lack of XRD pattern is evidence for a non-crystalline metal-organothiol. The exact chemistry of the new materials remains to be elucidated but metal precipitation via a biogenic organothiol is a potentially effective approach to the remediation of aggressive metal wastes.     

Structure, partial elemental composition, and size of Thiopedia rosea cells and platelets

The phototrophic purple sulfur bacterium Thiopedia rosea forms multicellular, gas-vacuolate, regular, flat aggregates (platelets, sheets) held together by slime. Platelets found in eutrophic water consisted of slime (85% of the total wet volume) and 16 cells, while the gas-filled vacuole occupied 44% of the volume of a single wet cell. Individual platelet cells contained central spindle-shaped gas vesicles (which together constitute the cell's gas vacuole), intracytoplasmic membrane vesicles (chromatophores), and peripheral sulfur globules. Cells were surrounded by a Gram-negative type cell envelope and were connected to neighboring cells of the same platelet by mostly unstructured slime. Cells contained detectable amounts of magnesium, phosphorus, sulfur, and potassium as determined by wavelength-dispersive X-ray microanalysis. The large size and relatively low slime density of the platelet, as well as the flat shape, could greatly decrease platelet sedimentation and so stabilize the position of T. rosea within its water column.     

The thermotropic phase behavior of cationic lipids: calorimetric, infrared spectroscopic and X-ray diffraction studies of lipid bilayer membranes composed of 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP)

The thermotropic phase behavior of lipid bilayer model membranes composed of the cationic lipid 1,2-di-O-myristoyl-3-N,N,N-trimethylaminopropane (DM-TAP) was examined by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. Aqueous dispersions of this lipid exhibit a highly energetic endothermic transition at 38.4 degrees C upon heating and two exothermic transitions between 20 and 30 degrees C upon cooling. These transitions are accompanied by enthalpy changes that are considerably greater than normally observed with typical gel/liquid--crystalline phase transitions and have been assigned to interconversions between lamellar crystalline and lamellar liquid--crystalline forms of this lipid. Both infrared spectroscopy and X-ray diffraction indicate that the lamellar crystalline phase is a highly ordered, substantially dehydrated structure in which the hydrocarbon chains are essentially immobilized in a distorted orthorhombic subcell. Upon heating to temperatures near 38.4 degrees C, this structure converts to a liquid-crystalline phase in which there is excessive swelling of the aqueous interlamellar spaces owing to charge repulsion between, and undulations of, the positively charged lipid surfaces. The polar/apolar interfaces of liquid--crystalline DM-TAP bilayers are not as well hydrated as those formed by other classes of phospho- and glycolipids. Such differences are attributed to the relatively small size of the polar headgroup and its limited capacity for interaction with moieties in the bilayer polar/apolar interface.     

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics

Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S⁰ globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label-free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10 h post-incubation [PI]), the beginning of sulfur consumption (20 h PI), and the end of sulfur consumption (40 h PI), respectively. We observed an increased abundance of the sulfide:quinone oxidoreductase (Sqr) proteins in 10 h PI indicating a sulfur production state. The periplasmic thiosulfate-oxidizing Sox enzymes and the dissimilatory sulfite reductase (Dsr) subunits showed an increased abundance in 20 h PI, corresponding to the sulfur-consuming state. In addition, we found that the abundance of the heterodisulfide-reductase and the sulfhydrogenase operons was influenced by electron donor availability and may be associated with sulfur metabolism. Further, we isolated and analyzed the extracellular sulfur globules in the different metabolic states to study their morphology and the sulfur cluster composition, yielding 58 previously uncharacterized proteins in purified globules. Our results show that C. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism in response to the availability of reduced sulfur compounds.     

X-ray diffraction analysis of internal wool lipids

Polarised optical microscopy (POM) and X-ray diffraction techniques were applied to intercellular lipids extracted from wool to study their structural arrangement in order to determine their role in the diffusion properties of wool fibre. Intercellular wool lipids (IWL) arranged as concentrated liposomes were shown to be a good intercellular lipid model, allowing their study by X-ray diffraction techniques. The results confirm that intercellular lipids of wool fibre are organised in a lamellar structure of 5.0–8.0 nm width, termed β-layer, which had been assumed to be lipids arranged as a bilayer. Structurally, internal wool lipids are distributed at least in two domains at low temperatures: an ordered phase made up of ceramides and free fatty acids (FFA) alone, arranged in crystal orthorhombic states separately, and a liquid crystal state when mixed together. At 40 °C there is a reversible phase transition produced by the melt of the crystal orthorhombic states, whereas the liquid crystal state remains until 65 °C.     

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.