Article pubs.acs.org/Langmuir
Dependence of Bicellar System Phase Behavior and Dynamics on Anionic Lipid Concentration Lauren MacEachern, Alexander Sylvester, Alanna Flynn, Ashkan Rahmani, and Michael R. Morrow* Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador A1B 3X7, Canada ABSTRACT: Bicellar dispersions of chain perdeuterated 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC-d54) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were prepared with the molar fraction of DHPC held fixed at 20% and varying amounts of DMPC replaced by the anionic lipid 1,2dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG). 2H NMR spectra were examined to assess the effect of added DMPG on mixture phase behavior and morphology. Quadrupole echo decay and quadrupole-Carr−Purcell− Mieboom−Gill echo train measurements provided information about slow motions contributing to echo decay in the high temperature phases. The spectra and quadrupole echo decay properties of DMPC-d54/DHPC (4:1) and DMPC-d54/DMPG/DHPC (3:1:1) were qualitatively similar. With increasing DMPG concentration, the transition between the magnetically orientable phase and the higher temperature phase became increasingly distinct, and the spectral shape and echo decay characteristics of the high temperature bicellar phase became increasingly similar to those of DMPC-d54 in the liquid crystalline phase. The observation that DMPG changes spectra in the orientable phase incrementally while increasing the distinction between the orientable and high temperature bicellar phases provides new insights into how DMPG influences bicellar mixture morphology.
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INTRODUCTION Magnetically orientable bilayered micelle particles1 formed by mixing short-chain and long-chain lipids have attracted much interest because of their potential utility for solid-state nuclear magnetic resonance (NMR) studies of membrane associated proteins.2−8 Nevertheless, mixtures of long-chain and shortchain lipids, particularly 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), also display a rich phase behavior and phenomenology beyond magnetic orientability,9−16 and such mixtures are important as lipid assemblies in which morphology and phase behavior are highly sensitive to the mixing properties of components with highly mismatched chain lengths. The interesting properties and phase behaviors of bicellar lipid mixtures reflect a competition between the entropic cost of separating the long-chain and short-chain components and the enthalpic cost of accommodating substantial chain-length mismatch with components mixed. The behavior of DMPC/ DHPC mixtures is sensitive to the molar ratio q = [DMPC]/ [DHPC]. For q greater than ∼2, the mixtures display at least three distinct ranges of behavior. At temperatures below the DMPC gel-to-liquid crystal transition temperature, the longchain and short-chain components are strongly separated, and such dispersions form bilayered disk micelles, or bicelles, with DHPC preferentially located in the highly curved and orientationally disordered disk edges and DMPC preferentially located in the more planar bilayer regions.17−20 In these structures, the DMPC chains are presumably ordered or extended, and the molar ratio q defines a relationship between planar area and confining perimeter that constrains available © 2013 American Chemical Society
area per lipid in the planar regions of the structure. As temperature increases through the DMPC gel-to-liquid crystal transition temperature, the small bicelle particles coalesce into larger assemblies21,22 in which the long-chain and short-chain components are still predominantly segregated. These structures, which display magnetic orientability, have been identified as worm-like or thread-like micelles that can aggregate to form a chiral nematic phase.12,23 The morphologies of bicellar mixture assemblies in this temperature range have been studied using a variety of techniques.12−14,21−27 This phase is also notably viscous.12,16,23,26,28,29 Above the temperature range over which magnetic orientation is observed, DMPC/DHPC mixtures are reported to form perforated lamellae.11,12,23,26,30 At higher temperatures, there is also evidence for DHPC migration into the more planar bilayer regions and for the presence of small, isotropically reorienting particles.9,15,31 Because bicellar mixture morphologies respond so strongly to changes in the mixing properties of their lipid constituents over a rather small temperature range, the effects of adding a third lipid species can provide interesting insights into the interactions between bilayer components. Anionic lipids are often components of surfactant lipid models used in NMR studies of hydrophobic lung surfactant proteins,32 and the characterization of bicellar mixtures containing a mixture of zwitterionic and anionic lipids is of particular interest because Received: December 27, 2012 Revised: February 25, 2013 Published: February 26, 2013 3688
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of their potential utility for such studies. There have been many reported studies of bicellar mixtures comprising DMPC, DHPC and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG).11,13,14,24,25,33−39 Struppe and co-workers investigated bicellar mixtures having 25% acidic lipids, either DMPG or dimyristoylphosphatidylserine (DMPS), and used them in studies of myristoylated peptides.33,34 Particular attention was paid to spectra of deuterated components under conditions of magnetic orientation and to the effect of varying pH on sample stability. Replacing 25% of the DMPC by DMPG was found to have little effect on the observed 2H NMR spectra. The effects of doping bicellar mixtures with small amounts of DMPG were studied using small angle neutron scattering (SANS) and polarized optical microscopy.11,13,14,24,25,35 Most of these studies were done with [DMPG]/[DMPC] ≤ 0.067. For lipid to water ratios of about 0.1 by weight, bicellar mixtures with [DMPG]/[DMPC] = 0.067 were reported to transform, at about 25 °C from a bicelle disk phase to a perforated lamellar phase.25 In a SANS experiment in which oscillating shear was used to induce alignment, DMPC/DMPG/DHPC mixtures with [DMPG]/[DMPC] = 0.02 and with [DMPG]/[DMPC] = 1 were found to transform directly from an isotropically reorienting small bicelle phase to a smectic lamellar phase in contrast to DMPC/DHPC mixtures, which passed through an intermediate nematic ribbon phase.14 In another series of studies,36,37 pulsed field gradient NMR was used to measure diffusion of molecules either through or along structures formed in the oriented phase of bicellar mixtures with [DMPG]/[DMPC] = 0.05 or with [DMPG]/[DMPC] = 0.01. By studying the diffusion of a triblock copolymer bilayer component, it was concluded that the morphologies of the oriented phase in the absence and presence of DMPG were ribbon and lamellar, respectively.37 A study of bicellar mixtures with [DMPG]/[DMPC] = 0.02 or with [DMPG]/[DMPC] = 0.1 using SANS, pulsed field gradient NMR, and 31P NMR confirmed that the oriented phase in bicellar samples containing DMPG is perforated lamellar. Despite the attention paid to bicellar mixtures containing DMPG, there does not seem to have been a systematic study of how the observed 2H NMR spectra and transitions change when the relative amounts of DMPC-d54 and DMPG are changed while maintaining a fixed molar ratio of long-chain to short-chain lipid component. In this work, 2H NMR and DSC have been used to investigate the effect of progressively replacing chain-perdeuterated DMPC (DMPC-d54) by DMPG in mixtures of DMPC-d54/DMPG/DHPC (4 − x:x:1). Spectral comparisons have been used to examine the effect of increasing relative DMPG concentrations on phase transitions in this bicellar mixture and on spectral characteristics that can provide information about properties of the resulting phases. Quadrupole echo decay measurements have been used to compare slow motions in the high temperature lamellar phase of the bicellar mixtures to those in the liquid crystalline phase of DMPC-d54 in multilamellar vesicles. The dependence of quadrupole-Carr−Purcell−Meiboom−Gill (q-CPMG) echo train decay rates on pulse spacings has been used to gain some additional information about the effect of adding DMPG on the distribution of correlation times for slow motions contributing to echo decay in the high temperature phase of the bicellar mixtures. Differential scanning calorimetry (DSC) has also been used to gain additional information about transitions in the DMPC/DHPC (4:1) mixture and to correlate observed spectral changes to changes in sample enthalpy.
Article
MATERIALS AND EXPERIMENTAL METHODS
Bicellar lipid mixtures were prepared with DMPC-d54/DMPG/DHPC molar ratios of 4:0:1, 3:1:1, 2.67:1.33:1, 2.5:1.5:1, and 2:2:1 at lipid weight fractions of 0.05 and 0.1. For comparison, a multilamellar vesicle dispersion of DMPC-d54 was also prepared. Lipids were obtained from Avanti Polar Lipids (Alabaster, Alabama) and used without further purification. Samples were prepared as described previously16 with total lipid weights in the range of 15−25 mg. For each sample, dry lipids were weighed and dissolved in 2:1 (v/v) chloroform/methanol. Solvent was then removed by rotary evaporation at 45 °C. Any residual solvent was removed by exposure to vacuum for several hours. Bicellar mixture samples were hydrated by using the required volume of 100 mM HEPES buffer (pH = 7) to wash the lipid dispersion from the walls of the flask. Samples were then vortexed and immersed in a sonicating bath at room temperature for 15 min. To enable further mixing and disruption of residual multilamellar vesicles, samples were then frozen in liquid nitrogen and thawed at 40 °C five times. Prepared samples were kept at approximately −8 °C prior to use. As is typical for bicellar mixtures,14 sample viscosity was higher at room temperature than at lower temperature. The multilamellar vesicle dispersion of DMPC-d54 was prepared without sonication or freeze−thaw cycling. While added salts can increase magnetic orientability in bicellar samples,40 the focus of this work was on phase behavior and lipid dynamics. For reasons discussed previously16 and to facilitate comparisons with previous studies of bicellar phase behavior,12,16 additional salts were not used for the preparation of bicellar dispersions in this work. After preparation, samples were transferred to an 8 mm diameter NMR tube with a volume of ∼400 μL. Deuterium NMR spectroscopy and quadrupole echo decay measurements were carried out using a locally assembled spectrometer and a 9.4 T superconducting magnet in which the 2H Larmor frequency is 61.4 MHz. Spectra were acquired using a quadrupole echo sequence41 with π/2 pulses of length 4.25−4.5 μs. For spectroscopy, the separation between π/2 pulses in the quadrupole echo sequence was 35 μs, and 2000−3000 transients were averaged. The repetition time for aquisition was 0.9 s. Oversampling42 was used to obtain effective dwell times of 4 μs in the free induction decays that were then Fourier transformed to obtain spectra. For quadrupole echo decay measurements, the dependence of quadrupole echo amplitude on the time, 2τ, between the first π/2 pulse in the quadrupole echo pulse sequence and the refocusing of the echo, was obtained by collecting echos for pulse separations varying between τ = 35 μs, and τ = 200 μs. For each echo, 800−1000 transients were averaged. Bloom and Sternin43 demonstrated that the effect of pulse timing on the decay of a quadrupole-Carr−Purcell−Meiboom−Gill (qCPMG) echo train can be used to detect contributions to echo decay from lipid bilayer motions with correlation times that are too long too contribute to motional narrowing of observed 2H NMR spectra. There have since been some additional applications of qCPMG to obtain information about slow motions contributing to quadrupole echo decay in deuterated lipid bilayers.44−46 The q-CPMG pulse sequence ([π/2]x−τ−[π/2]y−{2τ−[π/2]y}n−1) results in the formation of echoes at times 2nτ following the initial pulse. For very small values of τ, decay of the echo train decay is insensitive to modulation of the quadrupole interaction by slow motions. As τ is increased, contributions to echo decay from progressively slower motions are reintroduced, and it is possible to obtain information about the distribution of such motions.46 For this work, q-CPMG trains having up to 40 echoes were obtained for τ = 40 μs, τ = 50 μs, τ = 75 μs, τ = 100 μs, τ = 150 μs, τ = 200 μs, τ = 300 μs, τ = 400 μs, and τ = 500 μs. For longer values of τ, the number of echoes recorded, nmax, was constrained by the condition 2nmaxτ ≤ 8 ms. Echo amplitudes were measured from the average of 200−400 echo train transients. As described below, the echo train decays observed for large values of τ were nonexponential and consistent with the quadrupole interaction for each deuteron being modulated by fast motions, giving 3689
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a contribution to echo decay that was common for all deuterons, and at least one slow motion having different correlation times for different deuteron populations. It was previously found that the variation in qCPMG echo train decay behavior with τ could be approximated by summing signal amplitudes from a small number of deuteron populations with quadrupole interactions for each modulated by one slow motion, having a distinct correlation time for that population, and a contribution to echo decay from fast motions common to all deuteron populations. In this work, the q-CPMG echo train decays for a given sample and temperature were plotted as ln[A(2nτ)/A(2nτmin)] versus 2nτ, where A(2nτ) is the amplitude of the nth echo for a given τ and A(2nτmin) is the amplitude of the first echo recorded for the shortest τ. For a given sample at 52 °C, data for all nine values of τ, comprising 217 points, were simultaneously fit to a sum of echo train amplitudes corresponding to three deuteron populations having specified slow motion correlation times. The fitting parameters were the relative magnitudes (Ai) of the three populations, the second moments (ΔM2i) of the portion of the quadrupole interaction modulated by the slow motion for that population, and the contribution to echo decay rate from fast motions common to all populations (1/T2′ ). Because the fit depends only on relative populations, there are only six independent parameters to be fit simultaneously to the 217 data points. Fitting was done using a χ2 minimization routine implemented in the Octave programming environment.47 Uncertainties in the parameter values extracted from each fit were estimated by finding the change in each parameter required to increase χ2 by Δχ2 ≈ 7. For a χ2 minimization with six parameters, this provides an estimate of the 68% confidence level for each parameter.48 A sample of DMPC/DHPC (4:1) was also prepared for differential scanning calorimetry. The sample, containing 20.3 mg of DMPC, was initially hydrated in 240 μL of 100 mM HEPES buffer using the same procedure as for the deuterated samples. This suspension was then diluted by a factor of 100, and a 300 μL portion of the resulting suspension was placed into the sample cell of a TA Instruments Nano DSC (TA Instruments, New Castle, DE) where it was scanned, in both heating and cooling modes, five times at a rate of 1 °C/min. Baseline correction and integration were done using Origin 6.1 (OriginLab Corp. Northhampton, MA).
If the bilayer normal is oriented perpendicular to the applied magnetic field, the 2H NMR spectrum of a deuteron on a given chain segment is a doublet split by Δν =
2 3 e qQ SCD 4 h
(2)
where eQ is the deuteron quadrupole moment, eq is the component of the electric field gradient along its principal axis, and (e2qQ)/(h) = 167 kHz is the quadrupole coupling constant for carbon−deuterium bonds.50 For lipids with a perdeuterated chain, the 2H NMR spectrum of such an oriented bilayer is a superposition of doublets with intensity concentrated at the quadrupole splitting corresponding to the orientational order parameter plateau. If the sample morphology corresponds to a spherical distribution of bilayer normal directions, each doublet is spread into a Pake doublet with prominent edges at the quadrupole splitting corresponding to the bilayer normal being perpendicular to the magnetic field. Figure 1 shows 2H NMR spectra, at selected temperatures, for a DMPC-d54/DHPC (4:1) mixture and for a DMPC-d54/
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RESULTS AND DISCUSSION H NMR Spectroscopy. In partially ordered phases containing chain-deuterated lipids, axially symmetric acyl chain segment motions that modulate the angle θCD, between a given carbon−deuterium bond and the local symmetry axis for that motion, can be characterized by an orientational order parameter: 2
SCD =
1 3 cos2 θCD − 1 2
Figure 1. 2H NMR spectra at selected temperatures for (a) DMPCd54/DHPC (4:1) and (b) DMPC-d54/DMPG/DHPC (3:1:1). Lipid concentrations were ∼5% (w/v).
(1)
In liquid crystalline lipid bilayers, the symmetry axis is the bilayer normal, and the average is over the chain conformations accessed during the motion. For a given lipid acyl chain, the small orientational order parameter observed for methyl group deuterons reflects reorientation about the methyl axis in addition to large amplitude chain reorientation near the bilayer center. In general, orientational order parameter increases with increasing proximity of a methylene group to the headgroup end of the acyl chain due to the increasing constraint of chain motions. Near the headgroup end, chain motions are generally more restricted than near the far end of the chain, and the dependence of orientational order on position along the chain can be characterized by a plateau in the orientational order parameter profile.49−52
DMPG/DHPC (3:1:1) mixture. Both were prepared with lipid weight fractions of approximately 0.05. Comparison of the spectra in Figure 1 shows that, like DMPC-d54/DHPC (4:1), DMPC-d54/DMPG/DHPC (3:1:1) also progresses from isotropic reorientation, to anisotropic reorientation, to axially symmetric reorientation about an axis oriented perpendicular to the magnetic field. The unsplit line observed at low temperature reflects isotropic reorientation of small bicellar particles with a correlation time that is short as compared to the characteristic time scale of the 2H NMR experiment (∼10−5 s). The spectra in Figure 1 show that isotropic reorientation persists to a slightly higher temperature in the DMPC-d54/DMPG/DHPC (3:1:1) dispersion than in DMPC-d54/DHPC (4:1). Comparison with the spectra of a 3690
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different DMPC-d54/DHPC (4:1) sample, presented earlier,16 shows a similar difference. Around 20 °C, the spectra for both samples in Figure 1 indicate that the bicelles aggregate into larger structures in which the labeled lipid reorients aniostropically on the 2H NMR time scale. Except for the methyl deuterons at the acyl chain ends, spectral doublets corresponding to specific DMPCd54 methylene deuterons are not resolvable at 22 °C for either sample. Above 22 °C, the spectra for both samples in Figure 1a are increasingly characteristic of fast, axially symmetric reorientation about an axis preferentially oriented perpendicular to the magnetic field. The spectra in this range are superpositions of doublets with distributions of quadrupole splittings reflecting the orientational order parameter profile for chains in the oriented phase. The onset of axially symmetric motion about an oriented axis also appears to occur at a slightly higher temperature in the DMPC-d54/DMPG/DHPC (3:1:1) dispersion than in DMPC-d54/DHPC (4:1). Above 30 °C, the DMPC-d54/DHPC (4:1) spectra (Figure 1a) gradually become more characteristic of axially symmetric reorientation over a broader range of bilayer normal orientations. This is indicated by the spreading of intensity, for a given doublet, from the quadrupole splitting characteristic of bilayer normal magnetic orientation to smaller splittings corresponding to bilayer normals oriented at less than 90° from the magnetic field. While the distributions of intensity across the DMPC-d54/DHPC (4:1) spectra become characteristic of more randomly oriented bilayers with increasing temperature, it is difficult to identify a clear transition. The continuous nature of this change for some DMPC-d54/DHPC (4:1) dispersions has been noted before,16 but the extent to which this transition can be located by inspection does seem to vary among observations. A transition from the apparently oriented phase to the higher temperature phase does seem to be more apparent in the DMPC-d54/DMPG/DHPC (3:1:1) dispersion. Between 28 and 30 °C, individual doublets, particularly in the DMPC-d54/DMPG/DHPC (3:1:1) spectra of Figure 1b, become sharper and more clearly resolved, but there is little change in the distribution of quadrupole splittings. The higher temperature phase of bicellar mixtures is generally identified as a lamellar phase,9,12,13 but quadrupole echo decay observations imply significant differences between lipid dynamics in the high temperature bicellar mixture phase and the liquid crystalline phase of a DMPC-d54 multilamellar vesicle dispersion.16 The very sharp doublets observed in the high temperature spectra of Figure 1 are consistent with the very long quadrupole echo decay time reported earlier for the corresponding phase of DMPC-d54/DHPC (4:1).16 The doublet resolution in the high temperature spectra of DMPC-d54/DMPG/DHPC (3:1:1) is higher than is normally seen for multilamellar dispersions of DMPC-d54, spectra of which are shown below (Figure 2d). From 26 to 40 °C, the maximum splittings in the DMPCd54/DMPG/DHPC (3:1:1) spectra remain roughly constant, while those in the DMPC-d54/DHPC (4:1) spectra decrease. At 40 °C, the maximum DMPC-d54/DMPG/DHPC (3:1:1) splitting is about 23% higher than the corresponding DMPCd54/DHPC (4:1) splitting. This indicates that, at high temperature, the DMPC-d54 acyl chain orientational order is higher in the bicellar mixture containing DMPG. Figure 2 shows 2H NMR spectra, at selected temperatures, for three DMPC-d54/DMPG/DHPC mixtures prepared with lipid weight fractions of approximately 0.05. DMPC-d54 spectra that were previously presented16 have also been included for
Figure 2. 2H NMR spectra at selected temperatures for (a) DMPCd 54 /DMPG/DHPC (3:1:1), (b) DMPC-d 54 /DMPG/DHPC (2.67:1.33:1), (c) DMPC-d54/DMPG/DHPC (2:2:1), and (d) DMPC-d54. Lipid concentrations for the bicellar samples were ∼5% (w/v). The DMPC-d54 spectra were previously presented in a different format.16
comparison. The three mixed lipid samples differ in relative amounts of DMPC-d54 and DMPG but have the same molar fraction of DHPC. Samples in this series were also used for quadrupole echo decay measurements described below. In considering the observed behavior, it should be noted that chain perdeuteration will lower the main transition temperature of DMPC-d54 by ∼4−5 °C relative to that of normal DMPC or DMPG.53 Panel a of Figure 2 shows the DMPC-d54/DMPG/DHPC (3:1:1) spectra of Figure 1b, replotted for comparison. Panels b and c of Figure 2 show similar spectral series for DMPC-d54/ DMPG/DHPC (2.67:1.33:1) and for DMPC-d54/DMPG/ DHPC (2:2:1), respectively. Spectra for DMPC-d54/DMPG/ DHPC (2.5:1.5:1) (not shown) were similar to those in Figure 3691
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2c. Increasing the fraction of DMPG appears to slightly increase the temperatures at which the samples undergo the isotropicto-anisotropic transition and the transition from the oriented phase, at intermediate temperature, to the high temperature phase. At 26 and 28 °C, DMPC-d54/DMPG/DHPC (3:1:1) and DMPC-d54/DMPG/DHPC (2.67:1.33:1) are both in the orientable phase, and their spectra (Figure 2a and b, respectively) display similarly distributed quadrupole splittings but with more broadening of the spectral components in the sample with higher relative DMPG content. As discussed below, this broadening is consistent with the observation of relatively shorter echo decay times in these samples over this temperature range. In DMPC-d54/DMPG/DHPC (2:2:1) (Figure 2c), the distribution of spectral intensity at 28 and 30 °C still reflects sample orientation, but the doublets are even more strongly broadened. The transition from the more oriented phase to the high temperature phase is easily identified in both the DMPC-d54/DMPG/DHPC (2.67:1.33:1) and the DMPC-d54/DMPG/DHPC (2:2:1) spectral series (Figure 2b and c, respectively), and the spectra in the high temperature phases of both samples display distributions of intensity consistent with being superpositions of Pake doublets and thus characteristic of random bilayer orientation. These spectra are more similar to those in the liquid crystalline phase of the DMPC-d54 multilamellar dispersion (Figure 2d) than to those seen at lower DMPG concentration (Figure 2a). The 30 °C spectrum in Figure 2b appears to be a superposition of oriented phase and high temperature phase spectral characteristics, suggesting coexistence of these phases over a narrow temperature range. Such coexistence would imply that the transition into the higher temperature phase has some firstorder character. It is interesting to note that the high temperature spectra of all of the DMPC-d54/DMPG/DHPC mixtures have maximum splittings that are similar to those of the corresponding DMPCd54 spectra (Figure 2d). The distribution of intensity across the methylene portion of the high temperature spectra for DMPCd54/DMPG/DHPC (2:2:1) (Figure 2c) does differ slightly from the corresponding distributions for DMPC-d54/DMPG/ DHPC (2.67:1.33:1) (Figure 2b) and DMPC-d54 (Figure 2d). This reflects a broadening of the Pake doublets for the DMPCd54/DMPG/DHPC (2:2:1) relative to the samples at lower DMPG concentration. This relative broadening is also apparent in a comparison of the corresponding sharp methyl doublets near the spectral centers and reflects differences in transverse relaxation. Effects of DMPG on bilayer slow motions, which might affect relaxation, are described below. Bicelle phase behavior can be sensitive to lipid concentration, and a second series of samples was prepared with lipid weight fractions of approximately 0.1. Samples in this series were also used for q-CPMG echo train measurements described below. Figure 3 shows 2H NMR spectra for samples of DMPC-d54/ DHPC (4:1), DMPC-d54/DMPG/DHPC (3:1:1), DMPC-d54/ DMPG/DHPC (2.67:1.33:1), and DMPC-d54 at lipid fractions of 0.1. The spectra for DMPC-d54/DHPC (4:1) (Figure 3a) show a progression from isotropic reorientation at low temperature, through slow anisotropic reorientation to fast, axially symmetric reorientation between 24 and about 40 °C, and to the nominally lamellar phase at higher temperature. The observed behavior is simliar to that presented previously for a similarly prepared sample16 except that the orientation and the transition from oriented to lamellar is slightly more apparent in
Figure 3. 2H NMR spectra at selected temperatures for (a) DMPCd54/DHPC (4:1), (b) DMPC-d54/DMPG/DHPC (3:1:1), (c) DMPCd54/DMPG/DHPC (2.67:1.33:1), and (d) DMPC-d54. Lipid concentrations for the bicellar samples were ∼10% (w/v).
the current sample. The spectra for DMPC-d54/DMPG/DHPC (3:1:1) (Figure 3b) show the same progression of phases and are very similar to the spectra for the corresponding lipid mixture at a lipid concentration of 0.05 (Figure 2a) except for the persistence of orientation to slightly higher temperature. Similarly, the difference between spectra from the DMPC-d54/ DMPG/DHPC (2.67:1.33:1) sample in this series (Figure 3c) and those for the corresponding sample in the lower lipid concentration series (Figure 2b) is a shift of the observed transitions to slightly higher temperature in the sample with higher lipid concentration. Interestingly, the 34 °C spectrum in Figure 3c also shows coexistence of the oriented and higher temperature phases. The oriented phases of bicellar mixtures containing DMPG are reported to be perforated lamellae in contrast to those of 3692
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containing just DMPC and DHPC for which the oriented phase is reported to be ribbon-like.13,37,39 Nevertheless, the change in the phase behavior and in the progression of spectral shapes through the transitions, on going from DMPC-d54/ DHPC (4:1) to DMPC-d54/DMPG/DHPC (3:1:1) in Figure 3, is very subtle. Furthermore, the change in spectral shape on passing from the magnetically oriented phase to the higher temperature lamellar phase becomes more pronounced as the DMPG fraction is increased. It is not immediately clear how this can be reconciled with the implied qualitative difference between the oriented phases of bicellar samples with and without DMPG present. The abrupt transition between the magnetically oriented phase and the high temperature phase, in the bicellar mixtures containing DMPG, suggests that these phases are at least as distinct in the presence of DMPG as they are in the DMPC/DHPC mixtures. On the basis of small angle neutron scattering (SANS) experiments, the presence of DMPG was reported to promote one-dimensional swelling in the high temperature, presumably lamellar, phase.24 The same study reported that the addition of DMPG did not perturb basic morphology of the lipid component. This was interpreted as indicating a change in thickness of the water layer between bilayer sheets. While increased water thickness between bilayers might affect slow bilayer motions, the quadrupole splittings observed in 2H NMR spectra of chain-deuterated lipids in bilayers are not strongly affected by interbilayer spacing. The persistence of spectra characteristic of lamellar organization in the presence of increasing DMPG concentrations thus appears to be consistent with the finding, by SANS, that DMPG does not perturb morphology of the lipid component in the dispersion and does not rule out the possibility of concurrent swelling of the interbilayer water layer. Differential Scanning Calorimetry. While the transition from the magnetically orientable phase, at intermediate temperatures, to the higher temperature phase is quite apparent with DMPG present, the change in DMPC-d54/DHPC (4:1) spectral characteristics at this transition can be more subtle, as seen in Figure 3a and earlier work.16 Differential scanning calorimetry (DSC) can provide a useful complement to spectral studies of phase behavior. Figure 4 shows heating and cooling DSC scans for DMPC/DHPC (4:1). As noted above, the sample was diluted by a factor of 100 before being introduced into the DSC instrument. The scanned sample thus corresponds to a much smaller lipid to water ratio than was used for the spectroscopy samples. There have been a number of previous DSC observations reported for DMPC/DHPC mixtures,10,15,27,54 and the general shape of the heating endotherm reported here is very similar to some of those.10,15 On cooling, the scan shows a small exotherm near 29 °C, but most of the transition enthalpy is released in a large exotherm at 25 °C, which is just above the main liquid crystalto-gel transition temperature for DMPC bilayers. This suggests that complex phase behavior seen on warming these samples is not readily reversed by cooling. In comparing the DSC endotherm in Figure 4a to the spectra of DMPC-d54/DHPC (4:1) in Figure 3a, it should be noted that perdeuteration of DMPC-d54 lowers the gel to liquid crystal transition by about ∼4−5 °C. With roughly 80% of the lipids deuterated in the NMR sample, this implies that the temperature range of the observed endotherm would correspond to a range of ∼21−28 °C in the deuterated NMR sample. At the lower end of this range, the spectra in Figure 1a
Figure 4. Differential scanning calorimetry traces plotted as power versus temperature for DMPC/DHPC (4:1) suspended in excess water during (a) heating and (b) cooling at 1 °C/min.
and Figure 3a are characteristic of reorientation that is not axially symmetric on the 2H NMR time scale. At the upper end of this temperature range, the spectra are sharp doublets characteristic of fast axially symmetric reorientation of “melted” lipid chains. Thus, despite the high dilution of the DSC sample, the temperature range over which heat is absorbed corresponds closely to the temperature range over which the less dilute samples display significant changes in morphology and dynamics. The enthalpy change obtained by integrating this endotherm is 18.2 kJ per mole of DMPC. This is smaller than was found in an earlier DSC study of DMPC/DHPC mixtures15 but is still 80% of the enthalpy change, 22.6 kJ/ mol, expected for the gel-to-liquid crystal transition in DMPC.55 The enthalpy change associated with the phospholipid gel-to-liquid crystal transition is dominated by the energy required to structurally disorder the individual lipid molecules.56 It thus seems likely that the enthalpy absorbed by the bicellar mixture just below the onset of magnetic orientability is contributing primarily to the “melting” of DMPC chains. Quadrupole Echo Decay. Properties of the high temperature bicellar phase, and their responses to the incorporation of DMPG, can also be inferred from the characteristics of bilayer 3693
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As described elsewhere,16 the quadrupole echo decay rate for a given deuteron can be written as a sum of contributions from motions that modulate the quadrupole interaction felt by that deuteron. A given motion, labeled i, can be classified as fast or slow based on comparison of its correlation time, τci, with (ΔM2i)−1/2 where ΔM2i is the second moment of that portion of the quadrupole interaction modulated by that motion. The contributions to the quadrupole echo decay rate from fast or slow motions are, respectively, proportional to or inversely proportional to the correlation time for that motion. It was previously suggested that the observation of a very long quadrupole echo decay time, and thus a very low echo decay rate, for DMPC-d54/DHPC (4:1) in the high temperature phase could indicate either a decrease in correlation times for fast motions, as compared to liquid crystalline DMPC-d54, or an increase in correlation times for slow motions.16 The dependence of echo decay time on DMPG concentration suggests that, with increasing DMPG concentration, the spectrum of motions contributing to quadrupole echo decay in DMPC-d54/DMPG/DHPC approaches that in DMPC-d54 alone. Quadrupole-Carr−Purcell−Meiboom−Gill Echo Train Decay. To gain additional insight into how DMPG affects lipid dynamics in the bicellar mixtures, q-CPMG echo train decays were acquired, at selected temperatures, for the samples prepared with a lipid concentration of 0.1, spectra of which are shown in Figure 3. In the q-CPMG experiment, an initial quadrupole echo sequence with two π/2 pulses separated by τ is followed by additional π/2 pulses at intervals of 2τ. Echoes are formed at intervals of 2τ. For small pulse separations, the echo train decay reflects contributions from motions with short correlation times. Increasing the pulse separation reintroduces contributions to the echo train decay rate from motions with increasingly longer correlation times. For temperatures close to room temperature where, in the absence of active cooling, response of the probe temperature controller can be slow, radio frequency sample heating can be an issue for echo trains with small pulse separation. In bicellar lipid mixtures, the effect of pulse-separation-dependent heating is compounded, at low and intermediate temperatures, by the proximity to phase transitions where echo decay rates are most sensitive to temperature. At higher temperatures, though, where echo decay rates in the bicellar mixtures are less sensitive to temperature and where the temperature control system can more effectively compensate for small amounts of additional RF heating, the first echoes of a series of echo train decays collected with different values of τ should decay with 2nτ at a rate equivalent to that for a conventional quadrupole echo decay experiment. Figure 6 compares quadrupole echo decays at 46 and 52 °C to first echo amplitudes from q-CPMG experiments, including ones on DMPC-d54 alone, at the same temperatures. These comparisons illustrate the diffference between echo decay behaviors of DMPC-d54/DHPC (4:1) and DMPC-d54 alone, in the high temperature and liquid crystalline phases, respectively. They also confirm that echo decay times for bicellar samples containing DMPG fall between these limits for both lipid/water ratios used and regardless of whether decay is inferred from conventional quadrupole echoes or from the first echoes of qCPMG trains. Figure 7 shows quadrupole-Carr−Purcell−Mieboom−Gill echo train amplitudes for different pulse spacings obtained from four lipid mixtures at 52 °C. The increasingly nonexponential character of the echo train decay for longer pulse separations is
motions that contribute to quadrupole echo decay in deuterated samples. It was recently reported that quadrupole echo decay times in the high temperature phase of DMPC-d54/ DHPC (4:1) are much longer than typically observed for multilamellar dispersions of DMPC-d54 alone in the liquid crystalline phase.16 This was interpreted as indicating that the spectra of motions contributing to quadrupole echo decay were quantitatively different in the two cases. In the liquid crystalline phase, quadrupole echo decay is thought to reflect contributions both from slow collective motions, such as bilayer undulations, and from faster local motions such as reorientation about the bilayer normal and chain conformational changes. As noted above, raising DMPG fraction in the bicellar mixtures resulted in the 2H NMR spectra at high temperature becoming increasingly similar to those for the multilamellar dispersion of DMPC-d54 in the liquid crystalline phase. To investigate whether increasing DMPG fraction also modified dynamics in the high temperature phase of the bicellar mixtures, the dependence of quadrupole echo amplitude on pulse spacing was measured for the bicellar samples prepared at a lipid fraction of 0.05. Figure 5 shows quadrupole echo decay times
Figure 5. Temperature dependence of quadrupole echo decay time for (○) DMPC-d54/DHPC (4:1), (□) DMPC-d54/DMPG/DHPC (3:1:1), (◇) DMPC-d54/DMPG/DHPC (2.67:1.33:1), (△) DMPCd54/DMPG/DHPC (2.5:1.5:1), (▽) DMPC-d54/DMPG/DHPC (2:2:1), and (●) DMPC-d54. The DMPC-d54 data were previously presented in a different format.16
for the samples from which the 2H NMR spectra in Figure 1 and Figure 2 were obtained. For comparison, Figure 5 also shows data for DMPC-d54 that were previously presented in a different format.16 It can be seen that as DMPC-d54 is replaced by DMPG in these mixtures, the echo decay time in the high temperature phase of the bicellar dispersion approaches that of DMPC-d54 in the liquid crystalline phase. This suggests either that DMPG is directly counteracting the effect of DHPC on the spectrum of motions contributing to echo decay in the high temperature phase or that DMPG is modifying the spatial distribution of DHPC in a way that diminishes its effect on such motions. The echo decay times for the (3:1:1) and (2.67:1.67:1) DMPC-d54/DMPG/DHPC compositions both appear to have anomalously low values near 30 °C. As seen in Figure 2a and b, this corresponds to the transition from the oriented phase into the high temperature phase. The observed quadrupole echoes from which the anomalous echo decay times are obtained are particularly nonexponential reflecting the coexistence of two phases, with distinct echo decay behaviors, at this transition. 3694
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Figure 6. Quadrupole echo decays at (a) 46 °C and (b) 52 °C for (circle) DMPC-d54/DHPC (4:1), (triangle up) DMPC-d54/DMPG/ DHPC (3:1:1), (triangle down) DMPC-d 54 /DMPG/DHPC (2.67:1.33:1), (triangle left) DMPC-d54/DMPG/DHPC (2.5:1.5:1), (diamond) DMPC-d54/DMPG/DHPC (2:2:1), and (square) DMPCd54. Open symbols are for samples suspended at ∼5% (w/v), and solid symbols are for samples suspended at ∼10% (w/v). Uncertainties are comparable to symbol sizes.
characteristic of a system in which the quadrupole interactions of different deuterons are modulated by slow motions with different correlation times and by fast motions common to all deuterons. The dispersion of the echo train decays with pulse spacing reflects the distribution of slow motions. For a given pulse spacing, the initial decay is dominated by signal from deuterons whose slow motions have correlation times short enough to contribute to echo train decay for that pulse spacing. Once the signal from that population has decayed, the remaining signal, from deuterons with slow motions having correlation times that are too long to contribute to echo train decay at that pulse spacing, decays with a characteristic time determined by the fast motions common to all deuterons. These will include reorientation about the bilayer normal and trans−gauche isomerization. As pulse spacing is reduced, the threshold between deuterons with slow motions that can, or cannot, contribute to echo train decay moves to shorter correlation times, and the fraction of signal lost in the initial decay decreases. The echo train decays in Figure 7 display notable differences in vertical spread at large values of 2nτ. This indicates significant differences between the spectrum of slow motions in the high temperature phase of DMPC-d54/DHPC (4:1) and that in the liquid crystalline phase of DMPC-d54 alone. The DMPC-d54/DMPG/DHPC dispersions show intermediate behaviors. Modulation of the quadrupole Hamiltonian for a given deuteron by a particular motion i can be characterized by its correlation time, τci, and by ΔM2i, the second moment of that portion of the quadrupole interaction modulated by that
Figure 7. Quadrupole-Carr−Purcell−Mieboom−Gill echo-train amplitude decays at 52 °C for (a) DMPC-d54/DHPC (4:1), (b) DMPCd 54 /DMPG/DHPC (3:1:1), (c) DMPC-d 54 /DMPG/DHPC (2.67:1.33:1), and (d) DMPC-d54. Echo amplitudes for the train having echoes separarated by 2τ are plotted as ln[A(2nτ)/A(2nτmin)] versus 2nτ, where A(2nτ) is the amplitude of the nth echo and A(2nτmin) is the amplitude of the first echo in the train obtained with the shortest τ. Echo train decays were obtained for (○) 40 μs, (□) 50 μs, (△) 75 μs, (◇) 100 μs, (●) 150 μs, (■) 200 μs, (▲) 300 μs, (◆) 400 μs, and (▽) 500 μs. Uncertainties are comparable to symbol sizes. Solid lines show best fits using a model with populations of deuterons subject to one of three slow motions and a common fast reorientational motion as described in the text.
motion. For a q-CPMG echo train with echoes separated by 2τ, the contribution of this motion to the echo train decay rate is then43,44,57 ⎡ ⎛ τ ⎞⎤ τ R i = ΔM 2iτci⎢1 − ci tanh⎜ ⎟⎥ ⎢⎣ τ ⎝ τci ⎠⎥⎦
(3)
The nonexponential echo train decays seen in Figure 7 imply that the quadrupole interaction of each deuteron must be modulated by a superposition of at least one fast motion, on the 2 H NMR time scale, and one slower motion. The observation that, at large values of 2nτ, decays collected with different pulse separations have similar slopes but are displaced vertically implies that there must be some differences between the slow 3695
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motions experienced by different deuteron populations but that the decay due to fast motions must be similar for all deuteron populations. If it is assumed that the deuterons can be be divided into populations of magnitude Ai distinguished by a particular slow motion characterized by τci and ΔM2i, then the amplitude of an echo formed at 2nτ in a train of echoes separated by 2τ is45,46 A(2nτ ) =
N
⎛
⎧ ⎪
⎡
⎝
⎪
i
⎩
⎢⎣
∑ Ai exp⎜⎜−2nτ ⎨ΔM2iτci⎢1 − +
⎫⎞ 1 ⎪⎟ ⎬ ⎪⎟ T2′ ⎭ ⎠
⎛ τ ⎞⎤ τci tanh⎜ ⎟⎥ τ ⎝ τci ⎠⎥⎦
(4)
where 1/T′2 is the common contribution to the echo train decay rate from fast motions. In an earlier study, it was found that qCPMG echo train decays in deuterated dimyristoylphosphatidylserine (DMPS-d54) could be simulated using eq 4 by approximating the distribution of slow motions as a small number of discrete populations with correlation times chosen to span a reasonable range of slow motion correlation times.46 The solid lines in Figure 7 are fits of eq 4 to the observed qCPMG decays obtained by assuming that the distribution of slow motions can be approximated by three discrete populations with slow motion correlation times of τc1 = 2 × 10−5 s, τc2 = 3 × 10−4 s, and τc3 = 6 × 10−3 s. For a given sample, the decays for all τ values are fit simultaneously using a single set of parameters (1/T′2 along with Ai and ΔM2i for each of the three correlation times). The steps between τc1 and τc2 and between τc2 and τc3 are roughly equal on a logarithmic scale. These correlation times are, respectively, comparable to the shortest value of τ used, comparable to the longest value of τ used, and about an order of magnitude larger than the longest τ used. While this specific binning of slow motion correlation times is somewhat arbitrary, comparison of the parameters obtained by fitting to a specific set of correlation times does provide some qualitative insight into how changes in the spectrum of slow motions might account for changes in qCPMG behavior with sample composition. As in the earlier study, the amplitude of the free induction decay immediately following the initial pulse of the q-CPMG sequence, for a given sample, was estimated by extrapolating the initial decay of the echo train, for the shortest value of τ, back to 2nτ = 0. Despite the crudeness of using three distinct correlation times to approximate the distribution of slow motions in these samples, the fits shown in Figure 7 do capture the important aspects of how the q-CPMG echo train decays vary with pulse spacing for the four samples. Figure 8 shows the parameters corresponding to the qCPMG echo train best fit amplitudes shown in Figure 7. The contributions to echo train decay from fast motions common to all deuterons, represented by 1/T′2 (Figure 8c), show little dependence on sample composition. Similarly, second moments (Figure 8b) associated with slow motions in the two longest correlation-time bins also differ only slightly with sample composition. For the slow motions represented by the shortest correlation-time bin, the second moments of the modulated portion of the quadrupole interaction are similar for the three bicellar samples. The corresponding second moment for DMPC-d54 alone is significantly higher. It is the fractions of deuterons affected by motions in each of the three correlationtime bins (Figure 8a) that may display the most interesting
Figure 8. Parameters obtained by fitting the quadrupole-Carr− Purcell−Mieboom−Gill echo-train decays, in Figure 4, to a model having three populations of deuterons distinguished by the slow lipid motion contributing to quadrupole echo decay. In each panel, the bars, going from left to right, correspond to DMPC-d54/DHPC (4:1), DMPC-d 54 /DMPG/DHPC (3:1:1), DMPC-d 54 /DMPG/DHPC (2.67:1.33:1), and DMPC-d54 at 52 °C. Panel (a) shows the relative deuteron populations obtained by fits with slow motions constrained to have correlation times of 2 × 10−5, 3 × 10−4, or 6 × 10−3 s. Panel (b) shows fitted values of ΔM2i corresponding to the allowed slow motion for each population. Panel (c) shows the contribution to the echo decay rate from fast motions that are assumed to be common to all three deuteron populations.
dependence on sample composition. For DMPC-d54, the fraction in the longest correlation-time bin is about one-half of the fractions in each of the two shorter correlation time bins. For DMPC-d54/DHPC (4:1), nearly 70% of the population is associated with the longest correlation-time bin. The two samples containing DMPG display intermediate distributions 3696
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this phase might reflect a change in bilayer mechanical properties resulting from coupling of bilayer sides by DHPC aggregations. Increasing the fraction of DMPG in samples reduced the difference between quadrupole echo and q-CPMG decay behaviors of the bicellar mixtures and DMPC-d54. Two possibilities suggested are that this might reflect a competition between opposing effects of DMPG and DHPC on bilayer mechanical properties or that it might reflect a DMPGmediated modification of DMPC/DHPC mixing properties. In addition to providing some additional insights into the specific properties of DMPG-containing bicellar lipid mixtures, this work also suggests how systems, like bicellar mixtures, that are particularly sensitive to lipid mixing properties can be used to examine interactions between bilayer components. Despite reports that the magnetically orientable phase in DMPC/DMPG/DHPC mixtures is qualitatively different from the magnetically orientable phase in DMPC/DHPC mixtures, the observations presented here show that the series of observed spectra for a given sample and the motions responsible for echo decay in the high temperature bicellar phase change only incrementally as DMPG is added to the bicellar mixture. This work also shows that the transition between the magnetically orientable phase and the high temperature phase becomes more apparent as DMPC is added to the bicellar mixture. The current understanding of these systems is that DMPC/DHPC mixtures transform, on heating, from isotropically reorienting bicelles, through a magnetically oriented ribbon phase, to multilamellar vesicle phase, while DMPC/DMPG/DHPC mixtures transform directly from a bicelle phase to a phase containing aligned perforated lamellae.37 On the basis of the observations presented here, it appears that the current understanding may be incomplete or in need of some refinement.
with the sample having the higher of the two DMPG concentrations being more similar to DMPC-d54. The difference between the large quadrupole echo decay times observed in the high temperature phase of DMPC-d54/ DHPC (4:1) and the shorter decay times observed for multilamellar vesicle dispersions of DMPC-d54 in the liquid crystalline phase has been noted previously.16 At that time, it was suggested that this distinction might reflect a difference in the spectrum of slow motions. The comparison illustrated in Figure 8a appears to be consistent with that suggestion. In DMPC-d54/DHPC (4:1), the distribution of slow motions contributing to echo decay seems to be weighted toward longer correlation times. Adding DMPG shifts the distribution toward one more characteristic of DMPC-d54 with less weight in the bin corresponding to the longest correlation time. In the earlier study, it was suggested that the difference in slow motions between the bicellar and multilamellar vesicle samples, in their respective high temperature phases, might reflect damping of motions in the bicellar material resulting from coupling between leaves of a given bilayer by laterally aggregated DHPC in the form of pores. If so, the observations presented here suggest that the addition of DMPG mitigates, in some manner, the effect of DHPC on the spectrum of slow motions in the sample. One possibility is that DMPG might have an independent but offsetting effect on bilayer slow motion dynamics despite the fact that, in the absence of Ca2+, the phase behaviors and observed spectra of diacyl phosphatidylglycerol and diacyl phosphatidylcholine lipid bilayers are very similar.58,59 Indeed, there is some evidence58 that, in the liquid crystalline phase, the quadrupole echo decay time for DPPGd62, in the absence of Ca2+, is shorter than that of DPPC-d62. The reported tendency of DMPG to promote swelling of bicellar lamellae,24 presumably by altering interbilayer water layer thickness, might be related to its effects on slow motions detected by quadrupole echo decay in these samples. However, another possibility is that DMPG modulates the effect of DHPC in bicellar mixtures by modifying the mixing properties of the long- and short-chain lipids in these mixtures. The effect of DMPG on the spectra and phase behavior illustrated in Figure 2 and Figure 3 might be consistent with such an interpretation.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada. We thank Pamela Rennie and William Whelan for technical assistance particularly with differential scanning calorimetry.
CONCLUSIONS A series of bicellar lipid mixtures were prepared with DHPC, at a fixed molar fraction, as the short-chain lipid component and various mixtures of DMPC-d54 and DMPG as the long-chain lipid component. The effects of DMPG fraction on phase behavior and lipid dynamics, in the high temperature phase, were observed using 2H NMR spectroscopy and quadrupole echo decay measurements. Increasing the DMPG content was found to raise the temperature at which reorientation changes from isotropic to anisotropic and the temperature at which the orientable phase is transformed to the lamellar high temperature phase. Samples with higher DMPG content displayed greater degrees of spectral broadening in the oriented phase and increasing similarity to the spectra of liquid crystalline DMPC-d54 in the bicellar high temperature phase. Fitting of qCPMG echo train decays indicated that the long quadrupole echo decay times obtained from the high temperature phase of DMPC-d54/DHPC (4:1) reflect a shift of slow motion spectral density toward longer correlation times relative to the case for multilamellar vesicle dispersions of liquid crystalline DMPC-d54. One possibility to consider is that longer echo decay times in
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dx.doi.org/10.1021/la305136q | Langmuir 2013, 29, 3688−3699
