A Deuterium 2D NMR Exchange Study - American Chemical Society

6 Feb 2017 - Miranda L. Schmidt and James H. Davis*. University of Guelph, Department of Physics, 50 Stone Road East, Guelph, Ontario, Canada, N1G ...
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Liquid Disordered−Liquid Ordered Phase Coexistence in Lipid/ Cholesterol Mixtures: A Deuterium 2D NMR Exchange Study Miranda L. Schmidt and James H. Davis* University of Guelph, Department of Physics, 50 Stone Road East, Guelph, Ontario, Canada, N1G 2W1 ABSTRACT: Model membranes composed of two types of long chain phospholipids, one unsaturated and one saturated, along with cholesterol can exhibit two coexisting fluid phases (liquid disordered (Sd ) and liquid ordered (So )) at various temperatures and compositions. Here we used 1D and 2D 2H NMR to compare the behavior of multilamellar dispersions, magnetically oriented bicelles, and mechanically aligned bilayers on glass plates, all of which contain the same proportions of dipalmitoleoylphosphatidylcholine (DPoPC), dimyristoylphosphatidylcholine (DMPC), and cholesterol. We found that multilamellar dispersions and bilayers aligned on glass plates behave very similarly. These samples were close to a critical composition and exhibit exchange of the lipids between the two fluid phases at temperatures near the Sd to Sd −So phase boundary. On the other hand, when a short chain lipid is added to the ternary long chain lipid/cholesterol mixture to form bicelles, the phase behavior is changed significantly and the So phase occurs at a higher than expected temperature. In addition, there was no evidence of exchange of lipids between the Sd and So phases or critical fluctuations at the temperature where the bulk of the sample enters the two-phase region for these bicelles. It appears that the addition of the short chain lipid results in these samples no longer being near a critical composition.

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INTRODUCTION Biological membranes are composed of many different types of lipids, sterols, and proteins, making them complex and dynamic systems. One important sterol is cholesterol which affects the fluidity of the membrane. It is possible to obtain two coexisting fluid phases, the liquid disordered (Sd ) phase and the liquid ordered (So ) phase, in mixtures containing a long chain unsaturated phospholipid, a long chain saturated phospholipid, and cholesterol. Cholesterol will preferentially interact with the saturated lipid; as a result, coexisting cholesterol and saturated lipid-rich (So ) domains and cholesterol-poor, unsaturated lipidrich (Sd ) domains can be formed.1−4 Phase diagrams have been presented for ternary mixtures of 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol, obtained using solidstate deuterium NMR spectroscopy4,5 and fluorescence microscopy.5,6 These phase diagrams for DOPC/DPPC/ cholesterol contain a region of fluid−fluid phase coexistence and a line of critical points where the phase transition becomes continuous. The most significant difference between the Sd and So phases is that the saturated lipid hydrocarbon chains are much more highly ordered in the So phase than those in the Sd phase. The domains rich in cholesterol have a substantially increased hydrophobic thickness as compared with those of the normal fluid (Sd ) phase. The lipid raft hypothesis7 suggests that natural membranes which are rich in cholesterol may organize themselves into domains having significantly different hydro© 2017 American Chemical Society

carbon chain order to facilitate some membrane processes. Deuterium NMR can be used to study the phase behavior of model membranes, since the deuterium quadrupolar splitting is sensitive to changes in the molecular motion and orientational order of the phospholipid chains.8 The 2H quadrupolar splitting is9 ΔνQ =

3e 2qQ (3 cos2 θnB − 1)·SCD 4h

(1)

where eq is the electric field gradient at the H nucleus, eQ is the quadrupolar moment of the 2H nucleus, and θnB is the angle between the local bilayer normal (n) and the static magnetic field (B). The CD bond orientational order parameter is 2

SCD =

1 (3 cos2 θCD − 1) 2

(2)

where θCD is the angle between the relevant carbon−deuterium (CD) bond and the local bilayer normal which is an axis of symmetry for the molecular motions in a fluid bilayer phase. The angular brackets denote an average over motions which are fast on the spectroscopic time scale. As a result, the phase of the membrane can often be determined from the characteristics of the spectra. It is also possible to perform quantitative analysis of the moments of the spectra. Near a critical point for the onset of the Sd −So coexistence region in DOPC/DPPC/cholesterol Received: July 29, 2016 Revised: January 20, 2017 Published: February 6, 2017 1881

DOI: 10.1021/acs.langmuir.6b02834 Langmuir 2017, 33, 1881−1890

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the lipids.27,28 Commonly used bicelles made with 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2dicaproyl-sn-glycero-3-phosphocholine (DCPC) align such that the bilayer normal of the disk or perforated lamella is oriented perpendicular to the magnetic field, but biphenyl lipids, lanthanide ions, and some peptides can flip the orientation of the bicelles by 90°.29−32 Lipid mixtures can also be deposited onto glass slides which can be stacked to create a mechanically oriented sample.33 The orientation of the bilayer normal with respect to the magnetic field of the spectrometer can be controlled in this case. In this study, we compare lipid/cholesterol mixtures which exhibit similar phase behavior in multilamellar dispersions, bicelles, and oriented on glass slides using deuterium 2D exchange experiments. These mixtures have the same proportion of the long chain lipid and cholesterol. All of the samples showed Sd −So phase coexistence, and we determined whether lipids were undergoing critical fluctuations near the phase transition. The multilamellar dispersion and bilayers on glass slide samples have compositions that are near a critical composition and show critical behavior, whereas the bicelles are not near a critical composition and do not show any evidence of exchange, presumably due to the presence of the short chain lipid.

mixtures, a characteristic broadening of the 2H spectra is observed.10,11 Two-dimensional exchange experiments correlate the resonance frequency of a molecule at the beginning and end of a delay or “mixing time”. These experiments are sensitive to processes such as a change in conformation or molecular orientation12−16 or a change in phase17−21 in which a nucleus may switch from precessing at a resonant frequency of ωA in one molecular state to ωB in the other state or vice versa. In the case of 2H NMR, it is the quadrupolar splitting which changes between the different molecular environments and, of course, a change in the quadrupolar splitting results in a change in the precession frequency.12 Since the quadrupolar splitting of lipids in the So phase can be more than twice that of the same lipids in the Sd phase,4 the exchange of lipids between these two phases during the mixing time will result in off-diagonal signal intensity in a 2D 2H exchange experiment. Deuterium 2D exchange experiments can be used to study motions with correlation times of up to 1 s (determined by the longitudinal relaxation time T1 and/or the characteristic time for the decay of quadrupolar order T1Q).12,19,22 If we define δν as the quadrupolar splitting, and Δ(δν) as the difference in quadrupolar splittings in the liquid ordered and liquid disordered phases, then the time scale is on the order of 1/ Δ(δν), which is ∼10−6−10−4 s for the exchange of lipids between the Sd and So phases of a model membrane in which these phases coexist. The critical fluctuations observed in the composition of mixed lipid/cholesterol samples4−6,10,11,23−26 modulate the lipid 2H quadrupolar splittings and result in the appearance of off-diagonal signal intensity in 2D exchange spectra. These ternary mixtures can exhibit a line of critical compositions such that as the temperature is varied at any one of these compositions the sample may pass through a critical point where the distinction between Sd and So phases vanishes.4 At temperatures well above this critical point, the sample is in a single homogeneous phase with well-defined quadrupolar splittings determined largely by temperature and cholesterol concentration.4,6 As the temperature is lowered toward the critical temperature, the local composition of the sample (which is related to the order parameter for the critical behavior11) begins to fluctuate, resulting in fluctuations of the quadrupolar splittings. The correlation length for these fluctuations becomes longer, and the time scale for the fluctuations becomes slower as the critical point is approached.10 At the critical point, the correlation length and the time scale diverge and we observe the static two-phase coexistence at temperatures below the critical point. The exchange process we are trying to study using 2D NMR exchange experiments arises from these critical fluctuations. The small scale exchange of lipids across domain boundaries in the static two-phase coexistence region involves only a small fraction of the lipids and is not expected to contribute greatly to the off-diagonal signal intensity in these experiments. Model membrane samples for NMR studies can be prepared in different forms such as multilamellar dispersions, magnetically oriented bilayers (bicelles), and mechanically oriented samples (e.g., on glass plates). In multilamellar dispersions or powder samples, all orientations of the bilayer are equally likely. Magnetically aligned samples are formed by mixing long and short chain lipids. These bicelles spontaneously orient in a magnetic field due to the anisotropic magnetic susceptibility of

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MATERIALS AND METHODS

Lipids. The lipids used in this study, chain perdeuterated 1,2dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC-d54), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPoPC), and 1,2-dicaproylsn-glycero-3-phosphocholine (DCPC), were obtained from Avanti Polar Lipids Inc. (Alabaster, AL) in powder form and used without further purification. Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO). Powder Samples. Multilamellar dispersions of ternary mixtures were prepared by codissolving appropriate quantities of dry, powdered lipids and cholesterol in 100% ethanol in a round-bottomed flask. The solvent was then removed by lyophilizing overnight. The dry mixture was carefully scraped from the flask and weighed. A 50 mM phosphate buffer (pH 7.0) was added to the mixture at a ratio of 4:3 (lipid weight to buffer volume) which corresponds to roughly 30 waters/lipid and constitutes full hydration in all phases observed. The sample was mixed by alternating stirring by hand using a glass rod and gentle centrifugation until the mixture was homogeneous. Finally, the sample was transferred via centrifugation into a small glass tube (3 mm diameter) which was sealed using silicone to prevent any water loss during the experiments. For more details of this sample preparation technique, see Davis et al.4 Magnetically Aligned Samples. Bicelle samples were prepared in almost the same way that powder samples were prepared. In the case of bicelles, a short chain lipid is included in the mixture (giving a ratio q between the number of long chain lipids and short chain lipids). DMPC-d54, DPoPC, and cholesterol were weighed out as dry powders. Since DCPC is highly hygroscopic, a 2.5 mg/mL (DCPC/ethanol) stock solution was prepared and then an appropriate volume of the stock solution was added to the lipid mixture. Again, the mixture was lyophilized overnight to remove the solvent, and then, the dry mixture was scraped from the round-bottomed flask. In this case, 50 mM phosphate buffer (pH 7.0) was added such that the final ratio of buffer/total hydrated sample (w/w) was 60% buffer by weight. After mixing, samples were transferred into small glass tubes (3 mm diameter) which were sealed using silicone. Mechanically Aligned Samples. Glass slides (obtained from Marienfeld-Superior, Germany) were used to mechanically align bilayers. The appropriate amounts of the lipids and cholesterol were combined in ethanol. The components were codissolved, but the volume of solvent used was minimal. The solution was placed drop by drop onto the glass slides (dimensions of 4 × 25 mm2) using a pipet. 1882

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2D Data Processing. The pure absorption 2D deuterium exchange spectra were obtained using the process described by Schmidt et al.12 Two data sets were collected with different phases for the storage and reconversion 54.7° pulses. These two data sets produce a symmetric spectrum and an antisymmetric spectrum which can be summed to produce the pure absorption spectrum. The pure absorption spectrum correlates the observed spin’s precession frequencies, ωA and ωB during t1 and t2, respectively. The shape of these spectra depends on the molecular reorientation which occurs during the mixing time τm. When no slow dynamics occur in a system, the frequencies ωA and ωB are equal. In these cases, ωA does not change during the mixing time and as a result the signal is confined to a diagonal line where ωA = ωB. Frequency changes due to reorientations of the molecule during τm result in spectral intensity off the diagonal.37 When lipids are able to exchange between two or more environments characterized by different resonance frequencies on the time scale of τm, the result is a spreading out of the intensity from the diagonal of the pure absorption spectrum.13 Bruker TopSpin (Milton, ON) software was used to phase the spectra from the two data sets. For the first data set, the phase in the F2 dimension was set so that each 1D subspectrum was symmetric. The Fourier transform in F2 was performed, then the imaginary part of the spectrum was zeroed, and finally, the Fourier transformation in F1 was performed, resulting in a symmetric 2D spectrum. For the second data set, the phase in the F2 dimension was set so that each 1D subspectrum was antisymmetric. Again, the Fourier transform in F2 was performed, then the imaginary part of the spectrum was zeroed, and finally the Fourier transformation in F1 was performed this time with an added phase correction of 90° in order to get an antisymmetric 2D spectrum. After phasing, the two spectra were then summed using MATLAB R2016a (The MathWorks Inc., Natick, MA). A scale factor for the second spectrum was included, since the relaxation processes occurring during τm may not be the same for the two data sets.12,37,38 For the powder and glass slide samples, the scale factor was close to 1 for all τm values; however, for the bicelle samples, this scale factor was around 0.6. The offset for the coordinates was also set in order to ensure that the peaks of the two spectra were in the same position when adding. In some of the spectra, imperfect cancellation of the antidiagonal components of the two experiments results in a small net antidiagonal signal intensity. This is especially evident if the frequency domain signals are very sharp, as with the bicelle sample.

Each layer was allowed to dry at ambient temperature and pressure before another layer was added. The process was repeated until all of the sample was deposited on the slides. The slides were allowed to dry completely before being stacked and placed into a plastic U-shaped channel to provide stability and maintain alignment. The sample was put through a series of hydration/dehydration cycles at ambient pressure at ∼50 °C ending with hydration in order to align the bilayers. A drop of D2O was added to the water used for hydrating the samples in order to allow for the hydration level of the sample to be monitored. Small pieces of wet filter paper were placed on top of a clean glass slide before the sample was sealed in order to provide extra water. Although the hydration level changes slightly with time due to some small leakage of water from the sealed sample, under these conditions, as long as the spectrum at a given temperature remains constant, the sample can be considered to be fully hydrated. Samples were wrapped in plastic which was heat sealed. These samples did lose water over time; however, the hydration of the sample was monitored using the D2O peak and the shape of the 1D 2H spectra before and after 2D experiments to ensure that the sample remained fully hydrated for the duration of the experiments. Experimental Setup. 1D Experiments. The 1D 2H NMR experiments were performed using a quadrupolar echo pulse sequence8 on 500 and 600 MHz Bruker BioSpin (Milton, ON) spectrometers at 2H frequencies of 76.77 and 92.15 MHz, respectively. Home-made coils were used, and the 90° pulses were optimized and were kept as short as possible in order to minimize any artifacts. The 90° pulse lengths used were 2.5 μs at 76.77 MHz and 1.7 μs at 92.15 MHz, and the echo delay was 42.5 or 40 μs. The delay prior to acquisition was set such that some points before the top of the echo were recorded, the signal was manually phase corrected and shifted in the time domain in order to have one point at the top of the echo, and then the points before the top of the echo were removed. This is a critical process which results in symmetric spectra with a flat baseline.9 The temperature was calibrated using Pb(NO3)2,34,35 and the corrected temperatures are presented here. The chain-melting points of DPPC-d62 (occurring at 311 K) and DMPC-d54 (occurring at 292 K) were used as references for the calibrations. The chain melting transitions of the unlabeled lipids DOPC and DPoPC are at 25336 and 237 K (Avanti Polar Lipids, Alabaster, AL, USA), respectively. 2D Exchange Experiments. The 2D exchange experiments on the powder and bicelle samples were run on an 800 MHz Bruker BioSpin (Milton, ON) spectrometer at a 2H frequency of 122.84 MHz using a 90° pulse length of 2.75 μs. The 2D exchange experiments on the samples aligned on glass slides were run on a 500 MHz Bruker BioSpin (Milton, ON) spectrometer at a 2H frequency of 76.77 MHz. In this case, the 90° pulse length was 3.40 μs. The general form of the pulse sequence for the spin-1 exchange experiment is shown in Figure 1. Two data sets with different phases for the 54.7° storage and reconversion pulses were recorded, and then, the resulting spectra were added together to obtain the pure absorption line shape.12 Details of the data processing are discussed in the next section.

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RESULTS AND DISCUSSION Phase Coexistence in 1D Spectra. Phase diagrams of DOPC, DPPC, and cholesterol mixtures have been presented previously.4,5 These ternary mixtures exhibit two-fluid, Sd −So , phase coexistence over a broad range of temperatures and compositions. We have shown that ternary mixtures composed of unsaturated DPoPC, saturated DMPC, and cholesterol have phase behavior analogous to the DOPC/DPPC/cholesterol mixtures.39 Our motivation for comparing the three different types of model membranes (multilamellar dispersions, bicelles, or magnetically oriented samples and samples mechanically oriented on glass slides) was to explore the potential for using oriented samples to study critical fluctuations in these lipid mixtures and to study the effect of including membrane spanning peptides and/or membrane proteins on the critical behavior. 2H NMR spectra depicting the onset of the Sd −So coexistence region for each type of sample are shown in Figure 2. Figure 2a shows powder spectra at various temperatures for the multilamellar dispersion sample of 32:48:20 (DPoPC/ DMPC-d54/cholesterol). At temperatures well above the critical temperature for this composition, we observe the classic superposition of 2H powder patterns illustrated by the top spectrum obtained at 311 K. The sharp Pake doublet at the center, which has a quadrupolar splitting of approximately 3.9

Figure 1. Schematic diagram of the spin-1 exchange experiment pulse program which has the following form: 90° excitation pulse - t1 - 54.7° storage pulse - τm (mixing time) - 54.7° reconversion pulse - Δ - 90° pulse - Δ - t2. Note that Δ - 90° pulse - Δ is a quadrupolar echo segment which allows the 2H powder signal to be recorded with minimal distortion. Two data sets are recorded for 2H exchange ° and the experiments. For data set 1, the storage pulse is 54.7−y reconversion pulse is 54.7°y . For data set 2, the storage pulse is 54.7°−x and the reconversion pulse is 54.7x°.12,37,38 1883

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have relatively small splittings due to the large degree of motional freedom experienced by the chain positions close to the center of the lipid bilayer. For chain positions closer to the lipid−water interface, the quadrupolar splittings become quite large, being about 32 kHz for this particular sample composition and temperature. As the temperature is lowered, the quadrupolar splittings all tend to increase due to the reduction in motional averaging which occurs as the temperature decreases. For this sample composition, there is a critical point at approximately 294 K so that at temperatures slightly above this critical point the powder pattern line shapes begin to broaden due to critical fluctuations.4,11 At temperatures below the critical point, the sample enters a two-phase coexistence region and the 2H spectra reflect this by exhibiting two components, one characteristic of the cholesterol rich So phase which has larger quadrupolar splittings (up to about 50 kHz for the methylenes near the lipid−water interface) and the other characteristic of the cholesterol poor and unsaturated lipid rich Sd phase which has significantly smaller quadrupolar splittings (typically less than 32 kHz for the chain methylenes). Figure 2b shows the oriented sample spectra at various temperatures for bicelles made of 32:48:20 (DPoPC/DMPCd54/cholesterol) + DCPC with q = 3.5 ((mol DPoPC + mol DMPC-d54)/mol DCPC). Notice that the main transition from Sd to the Sd −So phase coexistence region is more than 5° higher in temperature for the bicelles (above 300 K) than it is for the powder sample (occurring at 294 K). The presence of the short chain lipid has an effect on the overall phase behavior of these model membranes.40,41 We have previously postulated that these bicelle systems are composed of both perforated lamellae and disk-shaped particles.39 Since the sample was prepared at ambient temperature where the lipid composition used is in the two-phase region, some of the particles may be physically isolated from the rest of the sample and have a higher than average cholesterol concentration, resulting in the small So contribution to the spectra even at high temperatures.39 In addition, there is a small isotropic contribution to the spectra which is larger at higher temperatures. Figure 2c shows oriented sample spectra for 32:48:20 (DPoPC/DMPC-d54/cholesterol) on glass plates at various temperatures. The small isotropic peak is due to the free labeled water in the sample. These samples are placed in the field such that the bilayer normals are parallel to the external magnetic field of the spectrometer, resulting in quadrupolar splittings that are twice those of the 90° edges of the powder pattern and the splittings of the bicelle samples where the bilayer normal of each bicelle is perpendicular to the field. For the samples aligned on glass slides, the transition from the Sd phase region to the Sd −So two-phase region occurs at a temperature about 2° higher than that for the multilamellar dispersions of the same composition. 2D Exchange Experiments. Two-dimensional 2H exchange experiments were performed on DPoPC/DMPC-d54/ cholesterol samples which exhibit Sd −So fluid phase coexistence as multilamellar dispersions, bicelles, and bilayers aligned on glass slides. In these spectra, we looked for evidence of lipids sampling the two different fluid phases. Diffusion processes, such as the lipids moving from one phase environment to the other, and compositional fluctuations which occur near a critical point can result in a spreading out of the intensity of the spectrum away from the diagonal.13 In liposomes and multilamellar dispersions, diffusion of the lipids over the curved

Figure 2. 2H spectra showing the onset of Sd −So phase coexistence in powder, bicelle, and glass slide samples of DPoPC, DMPC-d54, and cholesterol. (a) Powder 32:48:20 (DPoPC/DMPC-d54/cholesterol) at various temperatures. Collected at 76.77 MHz, 1024 scans. (b) 32:48:20 (DPoPC/DMPC-d54/cholesterol) + DCPC bicelles, q = 3.5 ((DPoPC + DMPC)/DCPC) at various temperatures. Collected at 92.15 MHz, 1024 scans. (c) 32:48:20 (DPoPC/DMPC-d54/cholesterol) oriented on glass slides with the bilayer normal parallel to the magnetic field at various temperatures. Collected at 76.77 MHz, 4096 scans. Note that the quadrupolar splittings for the sample aligned on glass slides are double those for the bicelles which are oriented with their bilayer normal perpendicular to the magnetic field.

kHz, arises from the deuterons on the chain terminal methyl groups. This feature sits on top of the powder patterns arising from the chain methylenes which have larger quadrupolar splittings. The chain positions close to the terminal methyls 1884

DOI: 10.1021/acs.langmuir.6b02834 Langmuir 2017, 33, 1881−1890

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Langmuir surface of small particles (those having radii smaller than or comparable to 4Dτm , where D is the lateral diffusion constant and τm is the mixing time) will lead to a change in the orientation of the local director, resulting in a change in quadrupolar splitting and off-diagonal signal intensity. Other motions, such as molecular reorientation via jumps between different orientations, can lead to ridges which occur on the offdiagonal of the spectrum.12 Multilamellar Dispersions. Figure 3 shows 2D 2H spectra from a powder sample of 32:48:20 (DPoPC/DMPC-d54/ cholesterol) at 292.2 K. This temperature is near the critical point for the phase transition from Sd to the Sd −So two-phase region where broadening is observed in 1D 2H spectra due to critical fluctuations.4 At very short mixing times, such as τ = 0.25 ms in part a, there is no evidence of exchange occurring between the lipid phases. On the other hand, at longer mixing times, such as τm = 5 ms, there is some broadening in the 2D spectra which is indicative of exchange. For display purposes, the spectra have all been scaled to the same amplitude. However, due to relaxation, as the mixing time is increased, the relative maximum intensity of the spectrum decreases. Relative to the τm = 5 ms mixing time, when τm = 0.25 ms, the maximum intensity is 2.8, while when τ = 40 ms the maximum intensity goes down to 0.6 (data not shown). In the spectra shown here, we have scaled the methyl groups to have the same amplitude and used the same contour levels for all mixing times to facilitate comparison. In Figure 4, the 2D spectra from the powder 32:48:20 (DPoPC/DMPC-d54/cholesterol) sample are compared at different temperatures, 311.0, 294.0, 292.8, and 288.2 K. At all temperatures, a mixing time of 5 ms was used and the methyl peaks were scaled to have the same amplitude. At 311.0 K, the highest temperature shown, the sample is well above its critical temperature and the spectrum diagonal is sharply defined, showing little evidence of any effects which might cause spreading of intensity away from the diagonal. At 294 K, the sample is very close to its critical temperature and there is a significant spreading out of the signal intensity away from the spectrum diagonal. This process is even more evident at 292.8 K where we are just able to see an indication of the formation of some liquid ordered phase domains. At lower temperatures, for example at 288.2 K which is well within the two-phase region, the critical fluctuations have ceased and the diagonal is again very sharply defined. Since the 2H NMR spectra of chain perdeuterated lipids have contributions from chain positions with widely differing degrees of chain order, there is a distribution of quadrupolar splittings. In 2D exchange spectra such as those shown here, the relevant time scale for the observation of off-diagonal signal intensity depends upon the difference in quadrupolar splittings of the labeled position in the different environments. For example, the methyl groups have quadrupolar splittings of 2−4 kHz in the Sd phase and of about 6−12 kHz in the So phase. This results in an exchange time scale of the order of ≈200 μs for the methyls. The methylene positions near the lipid−water interface have quadrupolar splittings closer to 20 kHz in the Sd phase and may be as large as 50 kHz in the So phase. This results in an exchange time scale of roughly 30 μs. For this reason, there may be some differential spreading out of intensity away from the diagonal depending on label position and the exchange time scale. For short mixing times, we can expect to see less spreading of intensity near the center of the spectrum (small

Figure 3. 2D 2H spectra on powder 32:48:20 (DPoPC/DMPC-d54/ cholesterol) at 292.2 K with different τm values: (a) 0.25 ms, (b) 5 ms, and (c) 10 ms. Contour level specifications: base, 50 000; increment factor, 1.5; number of contours, 8. Collected at 122.84 MHz, 64 scans.

quadrupolar splittings) than near the outside of the spectrum (large quadrupolar splittings). Although this sample is a multilamellar dispersion, by using a limited quantity of buffer (4:3 ratio lipid weight to buffer volume) while still maintaining full hydration and by avoiding the use of freeze/thaw cycles which tend to reduce particle size in lipid dispersions, the effects of lateral diffusion over the curved surfaces are minimized. To further illustrate this point, 1885

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we have performed the “selective inversion” experiment of Brown and Davis.42 In that article, it was shown that the origin of the orientation independence of the 2H spin−lattice relaxation time, T1, was probably due to the rapid lateral diffusion of lipids over the curved surfaces in the multilamellar dispersion. On inverting the center of the powder pattern line shape by using a selective 180° pulse, they observed that the resulting “hole” in the center of the spectrum was rapidly (within 5 ms) filled in by diffusion. Those experiments were performed at 51 °C on a 50/50 weight of lipid/volume of water sample of DPPC-d4/water (the lipid was specifically deuterated at the fourth carbon on both acyl chains). The results of the same experiment performed at a temperature of 294 K on our 32:48:20 (DPoPC/DMPC-d54/cholesterol) sample are shown in Figure 5. Of course, our present powder sample uses chain

Figure 5. Mixing time dependence of the difference signal intensity ΔA (following selective inversion of the center of the spectrum) for the 32:48:20 DPoPC:DMPC-d54:cholesterol sample at 294 K. The dashed line shows the fit to the data for the first 5 ms, while the solid line shows the fit to the data at long mixing times (from 100 to 500 ms). The initial decay time, T1s ≈ 20.2 ms, is comparable to the average spin−lattice relaxation for lipid methylenes, while the long time decay time, T1l ≈ 222 ms, is typical of the spin−lattice relaxation time for lipid methyl groups.

perdeuterated DMPC-d54, and the spectrum consists of many overlapping 2H powder patterns so that the interpretation of the results of this experiment is not as straightforward as that of Brown and Davis who used a specifically labeled lipid. In contrast to their experiment, after subtracting the spectrum with the selectively inverted central region from the spectrum obtained without the selective inversion, we observe an initial decay rate which is comparable to the normal average spin− lattice relaxation time (about 20 ms at 294.0 K). Brown and Davis observed that about 50% of the difference signal intensity had disappeared after only 5 ms, while we observe a decrease in difference signal intensity of only about 10−15% over that time scale. Although some parts of the sample must contain particles of a small enough radius that diffusion over their surface would result in an enhanced rate of decay, it is clear that most of the lipids are in much larger particles and show little or no effect due to lateral diffusion over the curved surface. Bicelles. Bicelles which exhibit Sd −So phase coexistence can be made with DPoPC, DMPC-d54, cholesterol, and DCPC.39

Figure 4. 2D 2H spectra on powder 32:48:20 (DPoPC/DMPC-d54/ cholesterol) with τm = 5 ms at different temperatures: (a) 311.0 K, (b) 294.0 K, (c) 292.8 K, and (d) 288.2 K. Contour level specifications: base, 50 000; increment factor, 1.5; number of contours, 8. Collected at 76.77 MHz, 128 scans. 1886

DOI: 10.1021/acs.langmuir.6b02834 Langmuir 2017, 33, 1881−1890

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Langmuir These bicelles align spontaneously with their bilayer normal perpendicular to the external magnetic field. As described above and seen in Figure 2b, in these bicelle samples, a small fraction of the sample is in the So phase at higher temperatures than expected on the basis of the phase behavior for the mixture without the short chain lipid. Figure 6 shows the 2D 2H exchange spectra for 32:48:20 (DPoPC/DMPC-d54/cholesterol) bicelles with q = 3.5 ((DPoPC + DMPC)/DCPC) at two temperatures and two mixing times. The transition of the bulk of the sample from the single Sd phase to the Sd −So coexisting phases occurs around 297.5 K. The 2D 2H exchange experiments were performed at 297.5 and 296.6 K. In Figure 6a and c, τm = 5 ms, and in Figure 6b and d, τm = 20 ms. As with the multilamellar dispersions, the methyl groups have been scaled such that they have the same intensity, and the same contour levels are used for comparison. In all cases, the spectra are very sharp and there is no evidence of exchange between the two phases or of critical fluctuations. Bilayers Oriented on Glass Slides. The phase behavior of mixtures of DPoPC/DMPC-d54/cholesterol bilayers oriented on glass slides follows the trend of the phase behavior of multilamellar dispersions of the same mixture. At high temperatures, the sample is in the Sd phase; as the temperature of the sample is lowered, the 1D spectra show broadening due to fluctuations near the onset of the Sd −So phase coexistence region, as shown in Figure 2. Figure 7 shows the 2D 2H exchange spectra for 32:48:20 (DPoPC/DMPC-d54/cholesterol) aligned on glass slides with the bilayer normal parallel to the external magnetic field at two temperatures and two mixing times. The onset of the two-phase region occurs at 296.0 K for the samples on glass plates, and at 293.7 K, the sample has welldefined Sd and So phases. In Figure 7a and c, τm = 5 ms, and in Figure 7b and d, τm = 10 ms. As with the spectra from other types of samples, the intensities of the methyl groups have been scaled to be equal, and the same contour levels are used for comparison. Since these spectra were taken with the bilayer normal aligned parallel to the static magnetic field, the splittings are twice as large as those which would have been obtained using a perpendicular orientation (analogous to the case with bicelles). This results in significantly reduced signal intensity for the methylene groups so that we have used somewhat lower level contours to illustrate the broadening around the diagonal. No such broadening is observed for the bicelle samples even at these lower contour levels. At 296.0 K which is near the transition, there is evidence of some broadening in the 2D spectra (Figure 7a and b), while at the lower temperature, 293.7 K, there are two distinct phases and the spectrum is sharper. This is the same result that is observed for the multilamellar dispersion samples, although the effect is more pronounced in the powder spectra than in the oriented spectra. This may be due to the small difference in the critical temperature or in the character of the critical behavior induced by the presence of the glass substrate or by minor differences in sample composition between multilamellar dispersions and mechanically aligned samples.

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CONCLUSIONS

There is evidence of exchange caused by critical fluctuations near the critical point for the transition from the Sd to Sd −So twophase regions in samples which have a critical composition. Mixtures of 32:48:20 (DPoPC/DMPC-d54/cholesterol) both as multilamellar dispersions and bilayers aligned on glass slides

Figure 6. 2D 2H spectra on 32:48:20 (DPoPC/DMPC-d54/ cholesterol) + DCPC bicelles. Two mixing times are shown at each temperature: (a) τm = 5 ms and (b) τm = 20 ms at 297.5 K and (c) τm 1887

DOI: 10.1021/acs.langmuir.6b02834 Langmuir 2017, 33, 1881−1890

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Langmuir Figure 6. continued = 5 ms and (d) τm = 20 ms at 296.6 K. The bicelles are oriented with their bilayer normals perpendicular to the magnetic field of the spectrometer. Contour level specifications: base, 50 000; increment factor, 1.5; number of contours, 8. Collected at 122.84 MHz, 64 scans.

show evidence of this exchange at temperatures near the transition. The exchange phenomenon is not present (or is much slower) at lower temperatures where the sample contains well-established domains of the Sd and So phases. On the other hand, there is no evidence of exchange of lipids between the Sd and So fluid phase domains in the bicelles. These bicelles contain the same molar ratio of DPoPC, DMPC-d54, and cholesterol as the other samples, but in order to form bicelles, the short chain lipid DCPC was required. The presence of the short chain lipid has an effect on the phase behavior of this model membrane. Although the bicelle mixture exhibits coexisting fluid phases, the location of a critical point depends sensitively on sample composition and in the presence of a fourth lipid component like DCPC the sample may not be near a critical point, meaning that we are not able to observe exchange between the phases which is detectable using 2D 2H exchange experiments. The persistence of a small fraction of bicelles in the So phase even to the highest temperatures studied is most likely due to the formation of some bicelle particles having compositions with higher than average cholesterol concentrations. As mentioned above, and discussed in Schmidt and Davis,39 preparation of the bicelles at ambient temperature may lead to some heterogeneity in composition of individual bicelles if they are not free to exchange molecules with one another (i.e., if some bicelles are physically disconnected or isolated from the bulk of the sample). The fact that most of the sample is able to undergo the phase changes in a manner analogous to that of the multilamellar dispersions indicates that either the bicelle particle sizes are much larger than the domains being formed (which must be on the order of microns or otherwise 2H NMR spectra would not show such clean phase separation) or that most of the molecules in the bulk system are able to exchange with one another at least on the time scale of the temperature changes made during the experiments. We anticipate that at a value of q = 3.5 and 60% water the sample will consist primarily of perforated bilayers but there are likely some disk shaped particles as well.39 A systematic investigation of bicelles containing DPoPC/DMPC/ cholesterol at different ratios and DCPC should be undertaken in order to determine whether there is a critical composition for these samples and whether they exhibit exchange phenomena in the same way as the multilamellar dispersions and mechanically oriented bilayers of ternary lipid/cholesterol mixtures. A more quantitative study of the effect of critical fluctuations on 2D exchange spectra could be undertaken using 2H NMR magic angle spinning (MAS) of bilayers on glass beads20,22 using specifically deuterated lipids. A model similar to that used to analyze the 2H NMR MAS sideband line widths10 could then be used to interpret the results. We have seen that oriented samples, whether mechanically oriented using glass plates or magnetically oriented using bicelles, display phase behavior which, although similar to that of multilamellar dispersions, does differ from that of unoriented samples in some respects. Before using such samples to study the effect of peptides and/ or proteins on the phase behavior and critical behavior of

Figure 7. 2D 2H spectra on 32:48:20 (DPoPC/DMPC-d54/ cholesterol) on glass slides. Two mixing times are shown at each temperature: (a) τm = 5 ms and (b) τm = 10 ms at 296.0 K and (c) τm = 5 ms and (d) τm = 10 ms at 293.7 K. This sample is oriented so that the bilayer normal is parallel to the magnetic field of the spectrometer. Contour level specifications: base, 10 000; increment factor, 1.8; number of contours, 8. Collected at 76.77 MHz, 512 scans. 1888

DOI: 10.1021/acs.langmuir.6b02834 Langmuir 2017, 33, 1881−1890

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Langmuir

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model membranes, it will be important to establish the behavior of the oriented lipid mixtures themselves.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

James H. Davis: 0000-0002-3522-0498 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, and the Ontario Ministry of Research and Innovation. M.L.S. was the recipient of an Alexander Graham Bell Canada Graduate Scholarship (2011− 2014) and an Ontario Graduate Scholarship (2014−2015). The authors would like to thank the staff of the University of Guelph NMR Centre for their help with the instrumentation.

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DOI: 10.1021/acs.langmuir.6b02834 Langmuir 2017, 33, 1881−1890