How Can a Lipid Be Distinguished From a Sugar
Int J Mol Sci. 2013 Apr; 14(four): 8148–8163.
Phospholipid Membrane Protection by Sugar Molecules during Dehydration—Insights into Molecular Mechanisms Using Scattering Techniques
Christopher J. Garvey
aneAustralian Nuclear Scientific discipline and Applied science Organisation, Locked Bag 2001, Kirrawee DC NSW 2232, Australia; E-Mail: ua.vog.otsna@kneb
Thomas Lenné
iiResearch School of Biological Sciences, the Australian National University, Canberra, ACT 0200, Commonwealth of australia; E-Postal service: ua.vog.bsta@ennel.samoht
Karen Fifty. Koster
threeDepartment of Biological science, The University of South Dakota, Vermillion, SD 57069, Us; Email: ude.dsu@retsok.nerak
Ben Kent
1Australian Nuclear Science and Engineering science Organisation, Locked Bag 2001, Kirrawee DC NSW 2232, Australia; Email: ua.vog.otsna@kneb
Gary Bryant
fourSchool of Practical Sciences, RMIT University, Melbourne, VIC 3001, Australia; E-Mail: ua.ude.timr@tnayrb.yrag
Received 2012 Dec fourteen; Revised 2013 Apr 3; Accepted 2013 Apr 9.
Abstract
Scattering techniques have played a fundamental role in our understanding of the structure and function of phospholipid membranes. These techniques have been applied widely to written report how different molecules (east.1000., cholesterol) can affect phospholipid membrane structure. However, at that place has been much less attending paid to the effects of molecules that remain in the aqueous phase. One important case is the office played past small solutes, particularly sugars, in protecting phospholipid membranes during drying or deadening freezing. In this paper, we present new results and a full general methodology, which illustrate how contrast variation minor angle neutron scattering (SANS) and synchrotron-based Ten-ray handful (pocket-size angle (SAXS) and wide angle (WAXS)) tin can be used to quantitatively sympathize the interactions between solutes and phospholipids. Specifically, we show the consignment of lipid phases with synchrotron SAXS and explain how SANS reveals the exclusion of sugars from the aqueous region in the item case of hexagonal II phases formed past phospholipids.
Keywords: cryobiology, anhydrobiology, X-ray scattering, contrast variation pocket-size bending neutron scattering, membranes, phospholipids, sugars
ane. Introduction
Cell membranes exist equally selective barriers between the jail cell cytoplasm, various intracellular compartments and the extracellular earth. They may facilitate ship or human activity as a variable permeability barrier for solutes and solvent (water) molecules. The ship properties of the membrane depend on the proteins that mediate the movement of well-nigh solutes and on the physical properties of the membrane lipids forming the bilayer in which the proteins are embedded [one,ii]. Maintaining the correct functioning of this permeability barrier is of disquisitional importance to the viability of the prison cell. Cellular aridity (caused past freezing and/or dry environments) causes changes in membrane lipid organization, which, in plow, bring about the loss of the normal semi-permeability of the membrane and, thus, death of the cell [3–six].
Generally, the transport of solutes and macromolecules across the cell membrane is much slower than that of water, and it is the water distribution that responds most quickly to changing environmental conditions, such as aridity—depending on the species and tissue, some of the water transport occurs through the lipids of the cell membrane, while more rapid improvidence occurs through specific h2o channels, called aquaporins [3,5]. Thus in deadening drying conditions and at temperatures above the formation of the burnished state where molecular mobility is abruptly arrested, i can assume that water potentials will come to equilibrium through water improvidence and that solutes volition not redistribute across the membrane appreciably during the drying process [4]. The effects of slow cooling are equivalent—when water ice forms in the extracellular solution the concentration of extracellular solutes—is increased, and because the membrane is relatively permeable to water, water may be drawn out of the cell much more than quickly than solutes may exist transported in. Equally further cooling occurs, the volume fraction of ice increases, further increasing the solute concentration in the non-frozen fraction, and more water is drawn out of the cell. Thus, the net effect of freezing on ho-hum timescales is to dehydrate and contract the book of the intracellular solution and is, in fact, similar to the furnishings of drying [6,seven]. Contempo work has likewise suggested that the effects of sugars on membranes are very strongly concentration-dependent [eight], with sugar lipid interactions at very low sugar concentrations, only exclusion from the membrane surface at higher concentrations [9].
one.one. Membrane Protection by Small Solutes
High concentrations of small carbohydrate molecules tin assist maintain the viability of cells during slow freezing or drying [vii,10–13]. Similarly, the integrity of model membranes may exist maintained by the presence of sugars during changes in hydration caused by freezing or thawing [14,15]. A much-cited caption of this issue proposes a specific interaction between lipid head groups and sugar molecules [xvi]. The interaction involves the replacement of water molecules at the lipid caput groups past sugar molecules; thus, this model is termed the water replacement hypothesis (WRH) [17,18]. The proposed interaction is very specific, and the WRH is heavily reliant on the specificity of certain pocket-sized solutes, such as trehalose and sucrose, equally protectants [16]. In improver, the WRH is only a qualitative model. An alternate explanation is the hydration forces explanation (HFE) [6,19], which frames the protective machinery of sugars in terms of the modulation of the interaction between membranes as they come into increasingly shut apposition equally jail cell volumes contract during drying or freezing. The effects of vitrification (the glass transition) are as well important at very low temperatures and/or hydrations [11,20,21], and the effect of glass germination on membranes has been quantitatively explained previously using the HFE [22]. Information technology is at present well established that the HFE can quantitatively explain the membrane protective effects of solutes at low and intermediate hydrations, although specific interactions (as proposed by the WRH) may exist of import in completely dry out systems.
1.two. Hydration Forces Explanation
At the heart of the HFE is the loss of jail cell book accompanying the loss of cell water, bringing membrane bilayers into close apposition, where they feel brusk-range repulsive hydration interactions, which can damage the membrane [23–26]. This model for the interactions between membrane bilayers has been experimentally verified past direct measurement of the forces between model membranes brought into shut approach. Curt-range forces between bilayers have been measured using a variety of experimental techniques, and the short-range repulsive hydration force has been clearly identified [27–thirty]. Sugars have been constitute to modulate this interaction, reducing the curt-range repulsive hydration interaction [31]. Equally the membranes come up into close repulsive apposition, the short-range hydration interaction becomes dominant, inducing a lateral compressive stress in the membrane. This compressive stress is responsible for transitions from the fluid lamellar phase (associated with normal membrane part) to other potentially deleterious phases, such every bit the gel and inverse hexagonal phases. The furnishings of the lateral compression in the membrane include demixing of membrane constituents, such as proteins and lipids, as well as phase transitions in the membrane [6,xix,32]. The consequence of sugars is to reduce the hydration force between membranes and, thus, the lateral compression in the plane of the membrane.
one.3. Phase Behavior of Phospholipids at Low Hydration
In order to examine these furnishings, we have undertaken a range of scattering studies on model systems consisting of phospholipids and simple sugars at a range of hydration levels. While real membranes are complex mixes of lipids and macromolecules, in order to make the problem tractable from a theoretical and experimental viewpoint, our work has focused on model lipid systems, which exhibit the aforementioned general trends as membranes. Lipids may be in a number of different phases, depending on hydration and temperature [33,34], and several of these are shown schematically in Figure 1. Under normal physiological conditions (total hydration), phospholipid membranes be primarily in the fluid lamellar phase (Effigy 1a). As water is removed at constant temperature, the phospholipids tin undergo a transition to the gel phase (Figure 1b), which occurs due to the lipids beingness compressed in the plane of the membrane, leading to freezing of the lipid tails. Nosotros have extensively investigated this transition in single lipid systems in the presence of sugars, controlling the moisture contents by equilibration with various relative humidities [35–38]. Depending on the phospholipid used, other membrane lipid phases may also be at low hydration. These include the inverse hexagonal phase (Figure 1c) and the ribbon phase (Figure 1d). Such not-bilayer phases have been studied extensively [39–46] and have been shown to be important in freezing and dehydration damage in biological systems [6,47–fifty]: the non-lamellar nature of these phases means that cell membranes, which undergo such transitions, can no longer role equally semipermeable barriers between the prison cell and its environment.
The lipid phases involved in freezing or desiccation-induced cellular damage: (a) the fluid lamellar phase consists of alternating layers of lipid bilayers (thickness, d B) and water (thickness, d w) and separation between head groups, d h. In the fluid lamellar phase, the tail bondage are packed rather randomly in the hydrophobic phase; (b) the gel phase is very like in geometry, with the deviation being a closer packing of head groups and extended frozen lipid chains; (c) the hexagonal stage causes loss of bilayer structure and is characterized by a hexagonal symmetry with ii characteristic repeat distances. Each hexagon has at its center a circular aqueduct of water projecting out of the folio surface; (d) the ribbon phase, where the unit cell is again characterized by two characterized by repeat distances. A ribbon-like channel formed by lipid head groups projects out of the folio.
The measurements reported here will cover a number of these phases and highlight the complementary type of information that can exist gained from a range of scattering techniques. The information presented has a particular emphasis on the localization and quantification of the sugar concentrations shut to the lipid head groups. However, the techniques described can exist applied to the study of whatever small molecules, which may interact with membranes and affect membrane structure.
2. Scattering Techniques
In order to exam quantitative models of the interactions of membranes with sugars [iv,eight,36,37], membrane structural parameters need to exist measured using scattering techniques. These techniques accept been applied with great effect to empathise the stage beliefs and structure of phospholipids on the nanoscale [51–55]. Not only are they diagnostic of the phase of the lipids, but they are able to excerpt structural details relating to the shape and spatial relationships between coexisting phases. In our example, they provide two very important structural parameters: the boilerplate chain-chain lateral spacing (which can be used to estimate the average headgroup spacing d h), and the bilayer repeat spacing d, which tin exist used to estimate d westward and d B (Figure 1a). This information can be supplemented by contrast variation SANS; this is a low resolution quantitative technique, which, combined with a skilful knowledge of the density, isotopic makeup and volume fraction of each component in the sample, allows us to sympathize the length-scale and nature of heterogeneities quantitatively.
2.1. X-Ray Scattering
Experimental measurements are made both on synchrotron [37] and lab-based X-ray sources [39]. The basic principle of the experiments is the same, where the scattered intensity is measured every bit a function of scattering angle relative to the incident axle [56]. In the cases discussed here, the scattered intensity is measured on an area detector backside the sample. As an consequence of scattering formalism and its theoretical treatment and besides so that measurements fabricated with different wavelengths of handful radiation may be compared, the scattered intensity is expressed in terms of the scattering vector, q, which represents the modulus of the change in momentum of the scattered radiation:
where λ is the wavelength of the scattered radiation and 2θ is the scattering bending relative to the incoming beam. The features, which are most prominent in the X-ray scattering patterns, are the peaks. The athwart position of these peaks is indicative of a periodic spacing within the sample, and they may be used to assign the lipid space group and measure out periodic spacings within the sample (eastward.one thousand., Figure 1). The characteristic distances, d, of the spacing may be calculated simply using the Bragg equation and the angular position of a first lodge diffraction summit:
where n is a positive integral number and is the gild of the reflection (n = 1 for commencement lodge) and λ the wavelength of the radiations. For the showtime lodge scattering tiptop, this can be rewritten as:
The samples considered here are "powder" type samples—i.e., they consist of many stacks of lipid bilayers oriented randomly, yielding isotropic scattering with respect to the incident axle [57], as shown past the ii-dimensional (powder) patterns, examples of which are shown in Figure 2. Figure 2a,b shows, respectively, a gel stage and a fluid stage. The gel phase (Figure 2a) is indicated by the actress reflections, equally well as the strength of the reflections relative to those in the fluid phase (Figure 2b). This is due to the increased lodge in the packing of the lipid chains in the gel phase relative to the fluid stage. Figure 2c shows the scattering pattern on an image detector from an inverse hexagonal phase, indicated by the not-linear spacing of the reflections, which index to and signal the important jail cell dimensions of hexagonal packing (Figure 1c).
Small bending X-ray handful (SAXS) patterns for DOPC (ane,ii-dioleoyl-sn-glycero-3-phosphocholine): (a) gel phase at −33 °C; (b) fluid phase at two.half-dozen °C; and (c) inverse hexagonal phase at 36 °C. In comparison with the fluid phase (b), the gel phase (a) has an extra reflection and stronger reflections. The inverse hexagonal stage (c) has more reflections at modest angles.
The pinhole scattering information is radially symmetric, and knowing the experimental geometry and wavelength of the scattered radiations, the data may exist radially averaged and plotted as intensity versus the scattering vector, q, equally shown in Figure iii. Each peak corresponds to a different order of Bragg scattering, which allows the determination of the relevant structural parameters for each phase. The WAXS radial averages are shown as insets in Figure 3. The wide peaks for the fluid and inverse hexagonal phases (Figure 3b,c) indicate that the chains are in the fluid configuration, while the sharper height in Figure 3a is indicative of the tighter packing in the gel phase.
Radial averages of the data in Effigy 2, plotted every bit intensity vs. q. The positions of the peaks determine the stage and relevant structural parameters. For the (a) gel and (b) fluid phases, the chief peak at low q determines the repeat spacing, d (Effigy 1a). As both samples are lamellar, the higher order peaks are the second, third, 4th and fifth social club reflections of this primary repeat spacing. For the (c) inverse hexagonal stage, the reflections yield d11 and dx. The wide-angle peaks (shown in the insets) yield the average chain-chain separation. The sharp superlative in (a) is indicative of the ordered gel phase, while the other two samples have chains in the fluid configuration, giving a wide peak.
On modern synchrotron X-ray scattering beam lines [58], we are able to measure out the positions of these peaks to good precision on timescales of the order seconds [37,38], also every bit using the tunable nature of the Ten-ray radiation to access different regions of reciprocal/q-space. This approach allows us to measure out the inter-bilayer spacing and the inter-lipid packing in bilayers (Effigy 3) using a single musical instrument configuration for a big number of samples and temperatures, likewise as allowing a report of the kinetics of the transitions with or without sugars [38]. Therefore, many measurements would be impractical using lab-based Ten-rays sources, and kinetic measurements would exist incommunicable.
The determination of the structural parameters for each phase at a range of hydrations and temperatures enables u.s. to link the HFE theory of inter-membrane interactions [four,26] to the structural changes caused by lateral compression in the membrane [37]. Additional information in the shape and relative intensities of college order peaks, due north > 1, allows the reconstruction of the electron density profiles. Calculations of the electron density profile establish that the electron density in the head group region is not altered by the presence of sugars in the aqueous phase [40]. This finding reinforces the decision that sugars are non preferentially located at the lipid head groups in partially dried samples.
two.2. Small Angle Neutron Handful
The technique of contrast variation SANS has particular power in this scientific problem. Although the technique inherently provides lower resolution than SAXS, its main advantage in this example is that the measurement provides quantitative information more than hands than SAXS, but also instruments are easily optimized for measurements over an extended q-range with a range of configurations/sample to detector distances. In common X-ray scattering measurements, the scattered intensity is measured as a function of minor angles around the management of the primary beam of neutrons, and the formalism is the aforementioned. For neutrons, however, rather than the heterogeneities existence due to variations in electron density, every bit is the case for X-ray scattering, heterogeneities are due to variations in nuclear backdrop of the constituents—i.e., the scattering length density (SLD). Isotopic exchange, generally of deuterium for hydrogen nuclei, allows u.s. to vary the scattering contribution of various sample components without changing the physics or chemical science of the organisation in an observable style. This isotopic commutation, or contrast variation, is a generic means to improve the data content of the low-resolution SANS technique [59,60]. In the case of crystallographic reconstruction of unit cells, it may besides be used to determine the phasing of the Fourier terms in the reconstruction of a unit cell [54]; however, conventional pinhole SANS instruments, which are optimized for neutron flux, have a larger spread of wavelengths and a smaller dynamic q-range [61] compared to typical SAXS beam lines [58]; so, the resolution is lower.
Nosotros have extended the work of Demé and Zemb [57] to include a range of sugars, hydrations and lipids. Regardless of the lamellar system used, we found that the presence of sugars leads to two aqueous phases in equilibrium with each other, but with quite different concentrations of saccharide: i aqueous stage betwixt the bilayers in the lamellae and some other, which does not contribute measurably to the SANS signal, in a bulk stage [35].
We have also applied contrast variation to non-lamellar lipids, hydrating with varying ratios of H2O:D2O. For SANS experiments, deuterated glucose is used in gild to enhance the neutron contrast—in our case, D6-glucose. Figure 4 shows an example of such a contrast variation series for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in backlog water at 45 °C, without (Figure 4a) and with (Figure 4b) deuterated glucose. Each experiment was conducted with 5 dissimilar H2O:DiiO ratios—changing the ratio of H2O to DiiO in the aqueous phase changes the relative scattering power (contrast) betwixt the aqueous phase and the membrane phase. In the sample containing just water and lipid, the contrast is due to the differences in handful light density (SLD) of the lipid and the aqueous phases. If the sample contains sugar, the scattering length density of the aqueous will too incorporate contributions from atoms other than those that reflect the composition of the solvent, i.eastward., the exchangeable hydrogens fastened to hydroxyl groups. The scattering curves on a log-log plot typically consist of a linear region, where the slope is close to −4, and one or more than peaks, which are equivalent to Bragg peaks found by x-rays, but broadened by the instrumental convolution of the SANS instrument [57].
Radially averaged small angle neutron scattering (SANS) data from (a) DOPE and (b) 0.5:1 glucose:DOPE for varying amounts of DiiO. The data show the typical course of a low q linear region and a peak due to the (one,0) plane of the HII phase at s higher q.
In order to make up one's mind the concentrations and locations of the sugars, the data in Figure four are analyzed past taking the square root of the scattered intensity at several different q-values, which lie in the linear region of the log-log plot. These values are then plotted as functions of theD2O book fraction, as shown in Figure 5. Each line represents the intensity values found at a single q-value (a vertical line through the handful curves in Effigy iv). The variation of intensity at a particular q-value from each data set with a different H2O:D2O ratio may be described by a quadratic equation, and a best-fit quadratic equation can exist determined for each q-value. Where these lines cantankerous, the axis is the point of zippo scattered intensity, called the match point: this is the point at which there is no contrast between lipid and the aqueous water channels in the HII phase, as shown schematically in the inset to Figure 5a. The minimum contrast, where the lines pass through nada, is represented by Schematic C. Since the isotopic ratio of water and the friction match point with a pure lipid phase are known, 1 can calculate the shift from the pure lipid value and, thus, the contribution of the D6-glucose in the channels to the match point. The position of the match point in the presence of glucose (b) allows the calculation of the sugar concentration in the water channels of the HIi stage. As shown in the figures, the lines all cross zero at the same betoken—this tells u.s. that the lucifer betoken is q-independent in the low q region, which is vital for the analysis to be valid [57].
Square root of intensity vs. D2O volume fraction for the data in Effigy 4. A schematic representation where the scattered intensity is proportional to the dissimilarity between the two phases is shown in the inset. In this instance, the scattering is due to the contrast, (or deviation in scattering length density)2, between the aqueous phase (various ratios of H2O:D2O:D6-glucose) and the lipid stage.
The match point is different for the pure lipid (Figure 5a) and the lipid with glucose (Figure 5b), since the composition of the solvent in the latter case has been altered by the Dhalf-dozen-glucose. Thus, from this data set, information technology is possible to summate the concentrations of sugar in the aqueous water channels of the H2 phase. Calculations reveal that the glucose concentration in the aqueous channels is lower than that in the bulk phase. This effect demonstrates that sugars are partially excluded from the HII stage water channels, implying that in that location are no dominant sugar-head group interactions and lending support to the HFE for the protective role of sugars during dehydration.
3. Discussion and Conclusions
Access to large scale facilities, in particular, synchrotron and neutron pocket-size angle scattering, has immune us to quantify factors relevant to the dehydration protection and cryo-protection of membranes past small solutes, specifically the altitude between lipid membranes and the spacing between lipid molecules packed in the membrane. Synchrotron X-ray scattering techniques provide a rapid method for measuring of import structural parameters and allow us to brand measurements on more samples and conditions than would exist possible using lab-based X-ray equipment. The resulting measurements have validated the hydration forces explanation (HFE) by directly relating the separation betwixt lipid bilayers and the separation between caput groups during the aforementioned measurement [37]. Contrast variation SANS allows the link betwixt the carbohydrate concentration in the lipid phase (lamellar or HII) to precise structural information from Ten-ray scattering.
Contrast variation SANS measurements on model systems indicates the exclusion of sugar molecules from between bilayers. While information technology is clear that this is an excluded volume issue, since larger molecules are excluded more effectively than smaller molecules [62], the quantification of this solute exclusion was not previously possible. SANS measurements take longer than synchrotron measurements (e.grand., on the order of tens of hours for the dissimilarity variation series shown in Figures iv and 5, whereas a single synchrotron measurements takes on the order of seconds), and while improvements in the neutron flux of modern SANS instruments may provide improvements on this situation, it will remain a depression throughput technique. Information technology is therefore very important to conduct complementary measurements using laboratory-based equipment to place regions of interest prior to attempting SANS measurements. For example, for the samples studied here, previous differential scanning calorimetry measurements have shown that the effects of sugars on the phase transition temperature saturate with increasing concentration [36], reducing the number of samples, which need to be studied using SANS.
Our investigations of the sectionalization of sugars in lipid systems have mostly concentrated on the lamellar systems, in particular, the fluid to gel stage transition. Still, it is known that not-lamellar phases, such as the changed hexagonal phase [41,42], are a critical component of dehydration and freezing damage [63]. Recently, nosotros have shown how the techniques described to a higher place may exist extended to studying the furnishings of sugars on non-lamellar phases, in particular, the changed hexagonal and ribbon phases (Figure 1c,d) [39,40]. These studies showed that the addition of glucose to a fully hydrated DOPE inverse hexagonal phase (Figures 2c and 3c) had no significant outcome on the structure of the stage and that glucose was (partially) excluded, similar to the results for the lamellar phases. However, these results too showed that the presence of glucose enhances the formation of the inverse hexagonal stage, which is in contrast with the observed power of sugars to limit damage to biological cells during dehydration. These results are currently undergoing further investigation.
In biological systems, transitions to non-lamellar phases would clearly lead to a loss in the continuity of the bulwark backdrop of the cell membrane. Thus, despite the challenges posed by these systems, X-ray diffraction and contrast variation SANS should provide a ways for relating the important structural parameters to the partitioning of aqueous carbohydrate molecules. Further experiments along these lines are currently underway.
The lower limit of hydration explored by the contrast variation technique, a sample equilibrated with 32% relative humidity, is greater than that experienced past many real membranes in extremely dry conditions. SANS measurements on materials at such low hydration levels are limited by the depression bespeak, which is nowadays at depression wet contents. 1 way of improving the signal to noise in SANS information from such samples is by deuteration of the lipid phase. Gains in signal volition be made due to the enhanced dissimilarity between aqueous and lipid phases, as well as the lower incoherent bespeak from the lipid stage [64], and this will allow us to obtain good quality measurements for systems at low hydration levels. Deuteration facilities are now becoming recognized as an integral tool in neutron scattering studies of biological systems (due east.g., Plant Laue Langevin, [65]; Australian Nuclear Scientific discipline and Applied science, organization, [66]; and the Centre for Structural Molecular Biology, Oak Ridge National Laboratory, [67]). Deuteration of lipid phases will likewise provide an invaluable tool in the phasing problem for the reconstruction of scattering length densities of orientated membrane systems and allow for higher resolution studies of the sugar concentration contour between lipid head groups [68]. These studies will provide a valuable complement to the electron density reconstructions of isotropic phases.
4. Materials and Methods
4.1. Small Angle Ten-Ray Scattering
SAXS/WAXS experiments were conducted at the Australian Synchrotron SAXS/WAXS beamline with λ = 0.827 Å and a sample to detector distance of 548 mm. Diffraction patterns were recorded on a 2D Dectris Pilatus 1M detector over a range of handful vectors from 0.073 to ane.96 Å−ane, covering the length scales of involvement for the primary repeat distance and the wide angle reflection. Exposure times were 1.9 southward. Samples were inserted into 1.5 mm quartz capillaries and sealed with epoxy resin. The pinhole handful data is radially symmetric and, together with the experimental geometry and wavelength of the scattered radiation, may be used to produce the radial boilerplate shown in Figure 3. The positions of the peaks may be used to measure the important inter lamellar and lipid packing spacings shown in Figure 1.
4.2. Small Angle Neutron Scattering
SANS measurements were performed on the QUOKKA beamline at the Bragg Institute (ANSTO, Australia) [61]. Samples were mounted in 0.ii mm path length Hellma cells. The incident neutron wavelength was v Å, with a resolution of ten% Δλ/λ (FWHM). Measurements were made at sample to detector distances of twenty.17 chiliad, 4.52 m and 1.24 yard, giving a combined q-range of 3.000 × 10−3 to 6.645 × 10−ane Å−1. Measurement count times at each distance were 30 min, 20 min and 10 min, respectively. Sample cells were sealed with lab wrap and positioned on a multi-sample changer side by side to a cadmium mask with a ane cm diameter cutout. Sample temperature was controlled using a circulating water bath. Measurements were made at 45 °C and were equilibrated for >1 h prior to measurement. Farther musical instrument specifications and details of the data reduction can be found online [69].
Neutrons were detected on a two-dimensional position sensitive detector. Afterwards correction for the detector response, the two-dimensional scattering patterns were checked to be isotropic at the 3 different camera lengths. Scattering patterns were normalized to the empty beam flux and sample thickness and appropriate backgrounds due to the empty cell and the isotropic incoherent signal, due mainly to 1H in the sample, subtracted.
4.3. Sample Preparation
Samples in all cases are prepared by exposing known molar ratios of lipids and sugars to a known humidity or by adding water gravimetrically. With increasing water content, d-spacings will increase, the moisture content at which the d-spacing does non expand further [cf Equations (two) and (iii)] and h2o partitions only into a bulk backlog water phase is termed excess hydration. Further details of preparing samples and their equilibration at different hydrations are given elsewhere [37]. For SANS measurements, the ratio of DtwoO:H2O was varied, and the samples packed to uniform density in quartz cells of a 200 μm path length with intendance taken non to shear the samples. Oriented structures volition produce anisotropic scattering patterns and make quantitative analysis difficult. X-ray scattering measurements were made on samples packed into thin-walled quartz capillaries of ane.five mm diameter.
Acknowledgments
Part of this research was undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia. We admit fiscal support to access the V4 SANS instrument at the Helmholtz Zentrum Berlin from the Access to Major Research Facilities Program, which is a component of the International Scientific discipline Linkages Program established under the Australian Government'southward innovation statement, Backing Australia'south Ability. Use of the ChemMatCARS Sector 15 at the Avant-garde Photon Source was supported by the Australian Synchrotron Research Program, which is funded past the Democracy of Australia nether the Major National Research Facilities Programme. ChemMatCARS Sector 15 is principally supported by the National Science Foundation/Department of Energy under grant number CHE-0535644. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Function of Science, Office of Basic Free energy Sciences, under Contract No. DE-AC02-06CH11357. During part of this enquiry, BK was the recipient of an Australian Found of Nuclear Science and Engineering postgraduate inquiry award.
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