Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.     

Dilatometric studies of isobranched phosphatidylcholines

Absolute apparent specific volumes have been obtained for phosphatidylcholine lipids with saturated, isobranched hydrocarbon chains with ni = 15 to 20 carbons, with an emphasis upon phase transition behavior, both equilibrium and kinetic. The temperature of the chain-melting transition extrapolates with increasing chain length to the melting temperature of polyethylene with a small odd/even alternation. There are also odd/even alternations in the volume of transition and in the hysteresis of the chain-melting transition, but with the odd and even reversed when compared with the larger odd/even alternation in the lower solid-solid transition that occurs in the longer chain ni lipids. A phenomenological picture is given for the coalescence of the two transitions for shorter ni lipids and this picture is used to sharpen the discussion of the kinetic mechanism of melting. A temperature-reversal experiment shows that the melting from the lowest temperature crystal or C phase to the fluid F phase does not proceed via the metastable gel G phase for 16i. The dilatometric results are combined with recent X-ray structural results for the C and G phases of 17i and 20i to deduce various structural information, including the hydration numbers and the volume of the headgroup, VH = 341 A3, which agrees very well with VH for straight-chain phosphatidylcholines. For the chain-melted F phase the assumption that the methylene volumes of the different ni lipids should be the same at the same temperature is used to obtain the volumes of the methylene and the methyl groups.     

Comparison of the effects of NaCl on the thermotropic behaviour of sn-1' and sn-3' stereoisomers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol

The phase behaviour of liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG) and the corresponding sn-3' stereoisomer (3'-DMPG) were studied by DSC as a function of NaCl concentration. The melting of the metastable gel phase to the liquid-crystalline phase was similar for both lipids. However, in the presence of salt and at 6 degrees C (T less than Tp) the gel phase of both stereoisomers of DMPG was shown to be metastable and a new phase nominated here as the highly crystalline phase was formed as the stable state. However, significant differences in the formation and melting of the highly crystalline phase were evident between the two polar headgroup stereoisomers. For 3'-DMPG in the presence of 300 mM NaCl the melting enthalpy of this phase is approx. 82 kJ/mol and the transition temperature about 11 degrees higher (at 33.6 degrees C) than for the gel to liquid-crystalline phase transition (25 kJ/mol at 23.0 degrees C). In the presence of 0.15-1.2 M NaCl at 6 to 10 degrees C the formation of the highly crystalline phase of 3'-DMPG is complete within 2 to 5 days, increasing [NaCl] facilitates the rate. For a 1:1 mixture of 1'- and 3'-DMPG the formation of the highly crystalline phase requires several weeks and melts at about 20 degrees higher than the gel phase (at approx. 40 degrees C). For 1'-DMPG partial conversion into the highly crystalline phase requires several months. For 3'-DMPG several intermediate phases appeared as endothermic peaks between the main phase transition temperature and the melting temperature of the highly crystalline phase. In contrast, for 1'-DMPG and the 1:1 mixture the subgel phase appears to be the only metastable intermediate phase. Different monovalent cations differ in their effect on the metastable behaviour.     

Pressure-induced elevation of phase transition temperature in dipalmitoylphosphatidylcholine bilayers: An electron spin resonance measurement of the enthalpy of phase transition

The application of 136 atm of helium pressure to an aqueous dispersion of dipalmitoylphosphatidylcholine increased the temperature of the primary phase transition at 40.4 ± 0.2 °C by 3.0 °C. The lower temperature pretransition at 30.5 ± 0.5 °C, thought to be due to phosphate headgroup reorganization, was increased by 1.7 °C. Addition of 4% dipalmitoylphosphatidic acid to the dipalmitoylphosphatidylcholine affected the phase transition in the head group region more than in the hydrocarbon chain region. The pressure and temperature data obtained, taken together with the literature value for the bilayer volume expansion during solid-fluid phase transition, and inserted into the Clausius-Clapeyron equation yield a ΔH value of 8.8 kcal/mole for this phase transition. This value is within experimental error of the ΔH value obtained from differential scanning calorimetry and serves to support the validity of the data and the experimental technique. Phase transition was observed by electron spin resonance measurement of the exclusion of the small spin label Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) from the solid domains of the bilayer. This result offers a possible explanation for the direct antagonism by high pressure of the effects of the inhalation anesthetics.     

The effect of potential-sensitive molecular probes on the thermal phase transition in dimyristoylphosphatidylcholine preparations

Differential scanning calorimetry (DSC) has been employed to determine the effect of five commonly employed extrinsic potential-sensitive probes on phase transitions of multilamellar suspensions of L-alpha-dimyristoylphosphatidylcholine (DMPC). At mol% values of less than five, the effect of these probes on the excess heat capacity curve in the vicinity of the gel to liquid crystal phase transition can be described by an equation based on the formation of ideal solutions in both phases. Even at up to 4 mol%, these dyes only moderately reduce the enthalpy change associated with this transition, but cause a marked decrease in the size of the cooperative unit parameter. The excess heat capacity profile for diS-C3-(5) is represented by the ideal solution equation, even at 12 mol%, whereas the suspensions with the other probes present at this level have profiles covering large temperature ranges. Multiple peaks appear at the higher levels for the negatively charged oxonols V and VI, and merocyanine 540, a result consistent with the presence of well-defined microdomains or even phase separation. The enthalpy change associated with the transition near 15 degrees C involving packing in the headgroup region is decreased significantly, indicating that the probes probably affect the lipid headgroup conformation, even at low levels. The cyanine probe diS-C3-(5) causes the heat capacity profile of small unilamellar vesicles to be transformed very rapidly into one similar to that of the vortexed lipid preparations, presumably by a dye-mediated vesicle fusion process, enhanced by the surface location of this probe. All our results are consistent with diS-C3-(5) being located on the surface of the bilayer in both phases, but a penetration of the other probes into the hydrocarbon region, at least in the liquid crystal phase.     

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.     

On the importance of the phosphocholine methyl groups for sphingomyelin/cholesterol interactions in membranes: a study with ceramide phosphoethanolamine

In this study, we have examined how the headgroup size and properties affect the membrane properties of sphingomyelin and interactions with cholesterol. We prepared N-palmitoyl ceramide phosphoethanolamine (PCPE) and compared its membrane behavior with D-erythro-N-palmitoyl-sphingomyelin (PSM), both in monolayers and bilayers. The pure PCPE monolayer did not show a phase transition at 22 degrees C (in contrast to PSM), but displayed a much higher inverse isothermal compressibility as compared to the PSM monolayer, indicating stronger intermolecular interactions between PCPEs than between PSMs. At 37 degrees C the PCPE monolayer was more expanded (than at 22 degrees C) and displayed a rather poorly defined phase transition. When cholesterol was comixed into the monolayer, a condensing effect of cholesterol on the lateral packing of the lipids in the monolayer could be observed. The phase transition from an ordered to a disordered state in bilayer membranes was determined by diphenylhexatriene steady-state anisotropy. Whereas the PSM bilayer became disordered at 41 degrees C, the PCPE bilayer main transition occurred around 64 degrees C. The diphenylhexatriene steady-state anisotropy values were similar in both PCPE and PSM bilayers before and after the phase transition, suggesting that the order in the hydrophobic core in both bilayer types was rather similar. The emission from Laurdan was blue shifted in PCPE bilayers in the gel phase when compared to the emission spectra from PSM bilayers, and the blue-shifted component in PCPE bilayers was retained also after the phase transition, suggesting that Laurdan molecules sensed a more hydrophobic environment at the PCPE interface compared to the PSM interface both below and above the bilayer melting temperature. Whereas PSM was able to form sterol-enriched domains in dominantly fluid bilayers (as determined from cholestatrienol dequenching experiments), PCPE failed to form such domains, suggesting that the size and/or properties of the headgroup was important for stabilizing sphingolipid/sterol interaction. In conclusion, our study has highlighted how the headgroup in sphingomyelin affect its membrane properties and interactions with cholesterol.     

Structure and thermotropic behavior of the Staphylococcus aureus lipid lysyl-dipalmitoylphosphatidylglycerol

We have characterized the structural and thermotropic properties of one of the most important lipids in the cell membrane of Staphylococcus aureus, lysyl-dipalmitoylphosphatidylglycerol (lysyl-DPPG). applying differential scanning calorimetry and small- and wide-angle x-ray scattering. Microcalorimetry revealed that under physiological conditions (phosphate buffer, 20 mM NaPi, 130 mM NaCl, pH 7.4), the synthetic lysyl-DPPG resembles the features of the parent dipalmitoylphosphatidylglycerol (DPPG) with respect to its melting behavior. However, in contrast to DPPG, lowering the pH did not significantly affect the main transition temperature (∼40°C) of lysyl-DPPG, which can be explained by its difference in protonization because of the lysine group. X-ray experiments yielded the first information on chain packing and morphology of lysyl-DPPG. We found that lysyl-DPPG forms an interdigitated lamellar phase below the chain-melting transition. This can be explained by the large headgroup area of lysyl-DPPG as a result of its charged lysine group, especially if the headgroup is arranged parallel to the bilayer plane. Additionally, lysyl-DPPG degradation products, such as lysine and free fatty acids, had significant influences on the melting behavior and led to a multicomponent melting transition. Our results indicate that the degradation of lysyl-DPPG takes place mainly during the hydration process but also depends on lipid storage time, pH, and thermal treatment. Detailed temperature-resolved experiments at pH 5.0 demonstrated the formation of a lamellar gel phase with tilted hydrocarbon chains and a ripple phase, coexisting with the interdigitated lysyl-DPPG bilayers.     

Headgroup hydration and motional order of lipids in lamellar liquid crystalline and inverted hexagonal phases of unsaturated phosphatidylethanolamine--a time-resolved fluorescence study

By the use of frequency domain cross-correlation fluorometry, the fluorescence lifetime of the water soluble probe 8,1-anilinonapthalene sulfonic acid (ANS) in aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and phosphatidylethanolamine transphosphatidylated from egg phosphatidylcholine (TPE) was measured. The orientational order parameter and rotational diffusion constant of the lipophilic probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were also determined in TPE dispersions. In agreement with a previous study on DOPE (Cheng (1989) Biophys. J. 55, 1025-1031), abrupt changes in both the order packing and rotational diffusion constant were found at the lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition of TPE. Owing to the subnanosecond resolution capability of this frequency domain fluorometric technique, the heterogeneous fluorescence decay of ANS was resolved into three distinct components with different decay lifetimes (tau's). They were 0 less than tau less than 0.5 ns, 2 less than tau less than 9 ns and tau greater than 15 ns. These lifetime regions were attributed to the partitioning of ANS into the bulk aqueous medium, the lipid/water interface and the lipid hydrocarbon region, respectively. These classifications of lifetime regions were further supported by the sensitivity of those lifetime components with the solvent isotopic shift of D2O. Similar to the changes of orientational order and rotational diffusion of lipophilic probe, the lifetime and intensity fraction of ANS associated with the lipid/water interfacial region declined abruptly at the L alpha-HII transition of both DOPE and TPE. This observation suggested that a dehydration of the lipid headgroup surface occurs at the L alpha-HII transition. This study provided evidence that both the lipid headgroup surface hydration and the lipid dynamics change drastically as a result of the macroscopic rearrangement of lipids at the L alpha-HII transition.     

On the propensity of phosphatidylglycerols to form interdigitated phases

We have determined the phase behavior of disaturated phosphatidylglycerols (PGs) of chain lengths n(CH2) = 14-18 at high pH and ionic strength using calorimetry, dilatometry, as well as x-ray diffraction. PGs with n(CH2) = 14 and 16 show thermotropic behavior similar to that of phosphatidylcholines (PCs). The area/lipid obtained in the gel phase is smaller than that reported for PCs despite the expected larger effective headgroup size. This can be explained by the tilting of the PG headgroup out of the bilayer plane, and we provide experimental evidence for a headgroup tilt transition. For distearoyl PG, we further find that the "usual" gel phase coexists with an interdigitated phase, which exhibits a transition from an orthorhombic into a hexagonal chain packing. The total amount of the interdigitated phase depends significantly on the temperature but is found to be largely independent of temperature equilibration time and different sample preparation protocols. Thus, the development of the interdigitated phase appears to be kinetically trapped. The formation of interdigitated phases in PGs at much smaller chain lengths than in PCs is of high relevance to interaction studies with antimicrobial peptides, as it provides a mechanism for the discrimination of membranes composed of different lipid species.     

An effective way to determine the melting curve

A new ‘two-phase’ simulation method with which to accurately predict the melting curve is proposed. The method requires, as a starting configuration, generating a two-phase coexistence state by employing a suitable ensemble. Examining a change in volume ratio of the two phases upon varying temperature (pressure) under a fixed pressure (temperature) allows us to determine the phase transition point. The Clausius–Clapeyron relationship can then be implemented as a guide to predict the nearby phase transition point. The method was applied to determine the solid–liquid phase boundary of the modified Lennard–Jones system. A better accuracy, as that achieved by the non-equilibrium relaxation method (Asano Y, Fuchizaki K. J Phys Soc Jpn. 2017;86:025001), was obtained but with much less computational cost.     

Formation of a new stable phase of phosphatidylglycerols.

Dilauroyl and dimyristoylphosphatidylglycerol (DMPG) form a more stable gel state when aqueous suspensions are incubated several days at low temperature (0-2 degrees C), pH 7.4 with 0.15 M NaCl. This gel state is characterized by a higher transition temperature and a higher transition enthalpy. The geometry of this gel state is distinguishable from the metastable gel state that forms rapidly upon hydration on the basis of its x-ray diffraction pattern. Infrared spectra in the CH2 scissoring region indicate that the stable gel phase of DMPG is also characterized by reduced reorientational fluctuations of acyl chains and increased interchain interactions. Analysis of vibrational bands due to ester carbonyl groups of DMPG suggests that the transition to a new gel phase is initiated by changes in the interfacial and/or headgroup region of the bilayer, most likely via formation of interlipid hydrogen bonds. The melting of the stable gel phase of DMPG is accompanied by a gross morphological change resulting in vesiculation.     

Hydrophilic/Hydrophobic balance determines morphology of glycolipids with oligolactose headgroups

The morphology of synthetic glycolipids with lactose oligomers (Lac N, the number of lactose units, N = 1, 2, 3) was studied in lamellar phase. By a systematic combination of differential scanning calorimetry and small- and wide-angle x-ray scattering experiments, the effects of hydrophilic/hydrophobic balance on their thermotropic phase behaviors were discussed. The dispersion of Lac 1 exhibited a crystalline-fluid phase transition, dominated by the strong van der Waals interaction between dihexadecyl chains. In the case of Lac 2, the hydrophilic/hydrophobic balance between the headgroup and the alkyl chains is shifted to the hydrophilic side, resulting in a gel-fluid phase transition with a decreased transition temperature and phase transition enthalpy. Different from the first two systems, the differential scanning calorimetry trace of Lac 3 showed much less remarkable peaks. The small- and wide-angle x-ray diffraction patterns did not reveal any transition in the chain ordering, suggesting that the correlation between the hexasaccharide headgroups is so strong that the melting of the alkyl chains was not allowed. Such dominant effects of the hydrophilic/hydrophobic balance on the morphology of Lac N lipids can be attributed to the small sterical mismatch between the alkyl chains and the linear, cylindrical oligolactose groups.     

The physical properties of glycosyl diacylglycerols. Calorimetric, X-ray diffraction and Fourier transform spectroscopic studies of a homologous series of 1,2-di-O-acyl-3-O-(beta-D-galactopyranosyl)-sn-glycerols.

We have synthesized a homologous series of saturated 1,2-di-O-n-acyl-3-O-(beta-D-galactopyranosyl)-sn-glycerols with odd- and even-numbered hydrocarbon chains ranging in length from 10 to 20 carbon atoms, and have investigated their physical properties using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectroscopy. The DSC results show a complex pattern of phase behaviour, which in a typical preheated sample consists of a lower temperature, moderately energetic lamellar gel/lamellar liquid-crystalline (L(beta)/L(alpha)) phase transition and a higher temperature, weakly energetic lamellar/nonlamellar phase transition. On annealing at a suitable temperature below the L(beta)/L(alpha) phase transition, the L(beta) phase converts to a lamellar crystalline (L(c1)) phase which may undergo a highly energetic L(c1)/L(alpha) or L(c1)/inverted hexagonal (H(II)) phase transition at very high temperatures on subsequent heating or convert to a second L(c2) phase in certain long chain compounds on storage at or below 4 degrees C. The transition temperatures and phase assignments for these galactolipids are supported by our XRD and FTIR spectroscopic measurements. The phase transition temperatures of all of these events are higher than those of the comparable phase transitions exhibited by the corresponding diacyl alpha- and beta-D-glucosyl glycerols. In contrast, the L(beta)/L(alpha) and lamellar/nonlamellar phase transition temperatures of the beta-D-galactosyl glycerols are lower than those of the corresponding diacyl phosphatidylethanolamines (PEs) and these glycolipids form inverted cubic phases at temperatures between the lamellar and H(II) phase regions. Our FTIR measurements indicate that in the L(beta) phase, the hydrocarbon chains form a hexagonally packed structure in which the headgroup and interfacial region are undergoing rapid motion, whereas the L(c) phase consists of a more highly ordered, hydrogen-bonded phase, in which the chains are packed in an orthorhombic subcell similar to that reported for the diacyl-beta-D-glucosyl-sn-glycerols. A comparison of the DSC data presented here with our earlier studies of other diacyl glycolipids shows that the rate of conversion from the L(beta) to the L(c) phase in the beta-D-galactosyl glycerols is slightly faster than that seen in the alpha-D-glucosyl glycerols and much faster than that seen in the corresponding beta-D-glucosyl glycerols. The similarities between the FTIR spectra and the first-order spacings for the lamellar phases in both the beta-D-glucosyl and galactosyl glycerols suggest that the headgroup orientations may be similar in both beta-anomers in all of their lamellar phases. Thus, the differences in their L(beta)/L(c) conversion kinetics and the lamellar/nonlamellar phase properties of these lipids probably arise from subtly different hydration and H-bonding interactions in the headgroup and interfacial regions of these phases. In the latter case, such differences would be expected to alter the ability of the polar headgroup to counterbalance the volume of the hydrocarbon chains. This perspective is discussed in the context of the mechanism for the L(alpha)/H(II) phase transition which we recently proposed, based on our X-ray diffraction measurements of a series of PEs.     

Lecithin bilayers. Density measurement and molecular interactions.

Density measurement are reported for bilayer dispersions of a series of saturated lecithins. For chain lengths with, respectively, 14, 15, 16, 17, and 18 carbons per chain, the values for the volume changes at the main transition are 0.027, 0.031, 0.037, 0.040 and 0.045 ml/g. The main transition temperature extrapolates with increasing chain length to the melting temperature of polyethylene. Volume changes at the lower transition are an order of magnitude smaller than the main transition. Single phase thermal expansion coefficients are also reported. The combination of X-ray data and density data indicated that the volume changes are predominantly due to the hydrocarbon chains, thus enabling the volume vCH2 of the methylene groups to be computed as a function of temperature. From this and knowledge of intermolecular interactions in hydrocarbon chains, the change in the interchain van der Waals energy, delta UvdW, at the main transition is computed for the lecithins and also for the alkanes and polyethylene at the melting transition. Using the experimental enthalpies of transition and delta UvdW, the energy equation is consistently balanced for all three systems. This yields estimates of the change in the number of gauche rotamers in the lecithins at the main transition. The consistency of these calculations supports the conclusion that the most important molecular energies for the main transition in lecithin bilayers are the hydrocarbon chain interactions and the rotational isomeric energies, and the conclusion that the main phase transition is analogous to the melting transition in the alkanes from the hexagonal phase to the liquid phase, but with some modifications.     

Conformational changes of the lecithin headgroup in monolayers at the air/water interface

The headgroup conformation of the phospholipid dipalmitoyl-glycero-phosphocholine (DPPC) in monolayers at the air/water interface has been studied by neutron reflection in the fluid like liquid-expanded (LE) and in the crystal like solid (S) phase. Information on the headgroup conformation in the two phases has been obtained by scattering contrast variation of the lipid monolayer using four differently deuterated species of DPPC: perdeuterated, chain perdeuterated, choline group perdeuterated and selectively headgroup deuterated. Since the measurements were done mainly on a subphase of null reflecting water (i.e. water scattering contrast matched to the air) there is no subphase contribution to reflectivity and the simplest one layer model can be employed for the data analysis, thus minimising the number of free parameters. A remarkable change of the headgroup orientation was observed between the LE and the S phase. We found that the phosphate-nitrogen dipole of the DPPC headgroup exhibits an in-plane orientation with respect to the monolayer in the LE phase but it assumes a more parallel orientation to the surface normal at lateral pressures above 30 mN/m (S phase). Moreover, this conformational change is accompanied by a significant alteration of the headgroup hydration.Abbreviations DPPC Dipalmitoyl-Phosphatidylcholine - DMPC Dimyristoyl-Phosphatidylcholine - DPPE Dipalmitoyl-Phosphatidylethanolamine - DMPE Dimyristoyl-Phosphatidylethanolamine - DMPA Dimyristoyl-Phosphatic Acid - DMPG Dimyristoyl-Phosphatidylglycerol Correspondence to: T M. Bayed     

Simultaneous and in situ analysis of thermal and volumetric properties of starch gelatinization over wide pressure and temperature ranges

A method for simultaneous and in situ analysis of thermal and volumetric properties of starch gelatinization from 0.1 to 100 MPa and from 283 to 430 K is described. The temperature of a very sensitive calorimetric detector containing a starch-water emulsion at a selected pressure is programmed to rise at a slow rate; volume variations are performed automatically to keep the selected pressure constant while the heat exchange rate and the volume are recorded. The method is demonstrated with a novel investigation of pressure effects on a sequence of three phase transitions in an aqueous emulsion of wheat starch (56 wt % water). The volume changes during the main endothermic transition (M), associated with melting of the crystalline part of the starch granules and a helix-coil transformation in amylopectin, but also with an important swelling, were separated into a volume increase associated with swelling and a volume decrease associated with the transition itself. Thermodynamic parameters for this transition together with their pressure dependencies have been obtained from four independent experiments at each pressure. The data are thermodynamically consistent, but are poorly described by the Clapeyron equation. The negative volume change of the slow exothermic transition (A) appearing just after the main endothermic transition (M) is small, spread out over a wide temperature interval, and occurs at higher temperatures with increasing pressures. This transition is probably associated with reassociation of the unwound helixes of amylopectin with parts of amylopectin molecules other than their original helix duplex partner. The positive volume change of the high-temperature, endothermic transition (N) with a small enthalpy change is probably associated with a nematic-isotropic transformation ending the formation of a homogeneous SOL phase (in the sense of Flory), and is also pushed to higher temperatures with increasing pressures. Knowledge of the state of wheat starch as a function of pressure and temperature is important in extruder processing. The data also provide a basis for the elliptic phase diagram for starch gelatinization. The method is easily adapted to determine similar data for other macromolecular materials.     

Calcium binding by phosphatidylserine headgroups. Deuterium NMR study.

The binding of calcium to headgroup deuterated 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine (POPS) was investigated by using deuterium magnetic resonance in pure POPS membranes and in mixed 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS 5:1 (m:m) bilayers. Addition of CaCl2 to pure POPS bilayers led to two component spectra attributed, respectively, to liquid-crystallin POPS (less than 15 kHz) and POPS molecules in the calcium-induced dehydrated phase (cochleate) (approximately 120 kHz). The liquid-crystalline component has nearly disappeared at a Ca2+ to POPS ratio of 0.5, indicating that, under such conditions, most of the POPS molecules are in the precipitated cochleate phase. After dilution of the POPS molecules in zwitterionic POPC membranes (POPC/POPS 5:1 m:m), single component spectra characteristic of POPS in the liquid-crystalline state were observed in the presence of Molar concentrations of calcium ions (Ca2+ to POPS ratio greater than 50), showing that the amount of dehydrated cochleate PS-Ca2+ phase, if any, was low (less than 5%) under such conditions. Deuterium NMR data obtained in the 15-50 degrees C temperature range with the mixed PC/PS membranes, either in the absence or the presence of Ca2+ ions, indicate that the serine headgroup undergoes a temperature-induced conformational change, independent of the presence of Ca2+. This is discussed in relation to other headgroup perturbations such as that observed upon change of the membrane surface charge density.     

Molecular response of the lipid headgroup to bilayer hydration monitored by 2H-NMR.

The effect of hydration on the conformation and dynamics of the phosphatidylcholine headgroup has been investigated by 2H-NMR measurements of liquid crystalline dioleoylphosphatidylcholine in multilamellar liposomes. Deuterium quadrupole splittings (delta nu Q) and spin-lattice relaxation rates (1/T1) were recorded for three selectively labeled headgroup segments (alpha, beta, and gamma) over the range of water/lipid mole ratios from 4 to 100. The smooth changes in delta nu Q and 1/T1 are found to essentially parallel each other and can be described by a single exponential decay function. Progressive hydration thus induces a concerted change in headgroup conformation together with an increase in its rate of motion (detected by delta nu Q and 1/T1, respectively). The enhanced mobility is partially due to a shift in the lipid phase transition temperature (as monitored by differential scanning calorimetry) and is furthermore attributed to an entropic contribution. It is concluded that the choline dipole becomes slightly raised in its average orientation into the aqueous layer and that the rate is increased at which the headgroup is fluctuating and protruding. The observed molecular changes can thus be accommodated within a model where the effective accessible headgroup volume expands with increasing hydration.     

Metastable behaviour of saturated phosphatidylethanolamines: a densitometric study

The specific volumes of saturated phosphatidylethanolamines having 12-18 carbon atoms per chain and the volume changes resulting from their temperature-induced phase transitions have been investigated using differential scanning densitometry. No dependence on acyl-chain length has been found for the molar volume changes associated with the Lc----L alpha transition, while the L beta----L alpha transition showed delta V-changes that increase with the length of the acyl-chains. The independence of acyl-chain length of the molar volume changes concomitant with the Lc----L alpha transition can be rationalized by the assumption that the volume changes result from the combined effects of hydration and acyl chain melting. An interesting corollary of the present studies is the disappearance of metastability at chain lengths of about 20-22 carbon atoms.