Liquid disordered–liquid ordered phase coexistence in bicelles containing unsaturated lipids and cholesterol

Liquid disordered–liquid ordered phase coexistence in bicelles containing unsaturated lipids and cholesterol

Biochimica et Biophysica Acta 1858 (2016) 619–626 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.else...

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Biochimica et Biophysica Acta 1858 (2016) 619–626

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem

Liquid disordered–liquid ordered phase coexistence in bicelles containing unsaturated lipids and cholesterol Miranda L. Schmidt, James H. Davis ⁎ University of Guelph, Department of Physics, 50 Stone Road E, Guelph, Ontario N1G 2W1, Canada

a r t i c l e

i n f o

Article history: Received 4 September 2015 Received in revised form 4 December 2015 Accepted 15 December 2015 Available online 17 December 2015 Keywords: Bicelles Cholesterol Liquid ordered phase Liquid disordered phase Nuclear magnetic resonance Model membrane

a b s t r a c t Magnetically orienting bicelles are often made by combining the long chain phospholipid 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC) with the short chain phospholipid 1,2-dicaproyl-sn-glycero-3phosphocholine (DCPC) in buffer. These bicelles orient with their bilayer normals perpendicular to the external magnetic field. We have examined the phase behaviour of DMPC/DCPC bicelles and the effects of cholesterol and the unsaturated phospholipid 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPoPC) as a function of temperature using static solid state 2H nuclear magnetic resonance spectroscopy. As expected, cholesterol has an ordering effect on the long phospholipid chains and this is reflected in the phase behaviour of the bicelle mixtures. Liquid disordered–liquid ordered, fluid–fluid phase coexistence is observed in DMPC/cholesterol/ DCPC bicelles with cholesterol mole fractions of 0.13 and higher. DPoPC/DMPC/cholesterol/DCPC bicelles also exhibit two fluid phase coexistence over a broad range of temperatures and compositions. Bicelles can provide a useful medium in which to study membrane bound peptides and proteins. The orientation parallel to the magnetic field is favourable for studying membrane peptides/proteins because information about the orientation of relevant molecular bonds or internuclear vectors can be obtained directly from the resulting 2H spectra. Lanthanide ions can be used to flip the bicelles to have their bilayer normals parallel to the external magnetic field. Yb3+ was used to flip the DPoPC/DMPC/cholesterol/DCPC bicelles while Eu3+ was found to be ineffective at flipping bicelles containing cholesterol in the present work. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Biological membranes are made up of many different lipids and proteins which are organized in a two dimensional bilayer structure. In many animal cell membranes cholesterol is a critical component because of its ability to affect the physical properties of the membrane [1]. Mixtures of lipids comprised of two long chain phospholipids, one with saturated acyl chains and one with unsaturated acyl chains, with cholesterol often exhibit the coexistence of two fluid membrane phases. These phases are the liquid disordered (‘d) and liquid ordered (‘o) phases and this phase coexistence can occur over a broad range of temperatures and sample compositions making them a useful mimetic for domain-forming membranes. Previous investigations of this kind of phase behaviour, specifically ‘d–‘o coexistence in ternary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), and cholesterol, using 2H Nuclear Magnetic Resonance (NMR) have been reported by Davis et al. [2–4] and Veatch et al. [5,6].

⁎ Corresponding author. E-mail addresses: [email protected] (M.L. Schmidt), [email protected] (J.H. Davis).

http://dx.doi.org/10.1016/j.bbamem.2015.12.016 0005-2736/© 2015 Elsevier B.V. All rights reserved.

2 H NMR is frequently used for studies of lipid phase behaviour because the deuterium quadrupolar splittings are sensitive to molecular motion and orientational order [7]. Static 2H spectra are significantly simpler for oriented samples than for powder samples in which all orientations occur with equal probability. Samples can be aligned either mechanically (by depositing lipid bilayers on glass plates for example) or magnetically (making use of the magnetic susceptibility of mixtures of long and short chain lipids) [8–10]. Magnetically aligned lipid bilayers (bicelles) are advantageous as a membrane mimetic since these oriented samples can give more signal for the same sample volume because there is no substrate needed to align them. Long chain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) combined with short chain 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) (often referred to as DHPC (1,2-dihexanoyl-sn-glycero-3phosphocholine)) forms bicelles which are often used either with soluble proteins for solution-state NMR experiments, or with membrane peptides or proteins which are embedded into the bicelles for solidstate NMR experiments [11–15]. Though DMPC/DCPC bicelles are most widely used, bicelle mixtures can be made from various types of long chain lipids and short chain lipids/detergents or modified lipids [16,17]. 31P and 2H NMR are used to characterize the phase behaviour of bicelles with long chain phospholipids of various lengths or types and different short chain phospholipids or detergents [11,14,15,

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18–21]. The morphology of the particles or structures forming these orientable phases is an interesting subject and has been the object of considerable discussion [14,20,22,23]. Details of the structures formed depend on the lipid composition, the long to short chain ratio, q, the water concentration and the temperature. For values of q near 3 and water concentrations similar to those used in this study it is generally felt that the orientable phase consists primarily of perforated bilayers but that there may be a population of disk shaped particles as well. The essential feature for the purposes of this study is that the structures orient well in the magnetic field and be large enough to support two phase coexistence with domains large enough to be studied by NMR. This requires that the particles be roughly 200 nm or larger in dimension. The effect of a polyunsaturated lipid (1-palmitoyl-2-linoleoyl-snglycero-3-phosphocholine (PLiPC)) and cholesterol on DMPC/DCPC bicelles has been investigated by Minto et al. [24]. They found that cholesterol increases the minimum alignment temperature, while the PLiPC decreases the minimum temperature for alignment of the bicelles. Cho et al. [25] reported two phase coexistence in POPC/DMPC/cholesterol/ DCPC bicelles using lateral diffusion measurements by 1H magic angle spinning NMR. Two fluid phase coexistence within aligned lipid samples is of interest for the investigation of peptides and proteins in these types of systems. In the present work, the phase behaviour of bicelles containing cholesterol is investigated and the coexistence of the ‘d and ‘o fluid phases is directly observed using static 2H NMR. In the next section we present the experimental procedures used to prepare and study these systems. This is followed by a description of the orientational order and phase behaviour in the presence of cholesterol and/or unsaturated phospholipids. We observe a broad range of ‘d–‘o phase coexistence. We also find that at high cholesterol concentrations in the DMPC/DCPC bicelles there is a significant fraction of the sample which is in an isotropic phase over the entire temperature range studied. However, the bicelles made with DMPC/DPoPC/DCPC and cholesterol possess a large ‘d–‘o coexistence region with little or no isotropic phase, making them more suitable for studies of peptide or protein partitioning between the ‘d or ‘o phases. We also find that there may be some bicelles formed which are physically isolated and unable to exchange molecules with the rest of the sample. This may affect their suitability for studies of partitioning between the phases. We conclude with a summary of the results and suggestions for the use of these mixed bicelles for studying membrane peptides and proteins. 2. Materials and methods Chain perdeuterated 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC-d54), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPoPC), and 1,2-dicaproyl-sn-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). Lanthanide salts were used to change the orientation of some bicelle samples; YbCl3 and EuCl3 were also purchased from Sigma Aldrich (St. Louis, MO). 2.1. Powder samples Multilamellar dispersions of lipids were prepared by mixing the appropriate quantities of the dry, powdered lipids and cholesterol in ethanol until completely dissolved in a round-bottomed flask. The solvent was removed by lyophilizing overnight. The dry mixture was then carefully scraped from the flask and weighed. 50 mM phosphate buffer (pH 7.0) was added at a ratio of 4:3 (lipid weight to buffer volume) and the mixture was stirred alternately by hand using a glass rod and gentle centrifugation until the mixture was homogeneous. The sample was transferred into a small glass sample tube which was sealed using silicone to prevent any water loss during the experiments. Details of this sample preparation technique are discussed by Davis et al. [2].

2.2. Bicelle samples Bicelle samples were made in a manner very similar to the powder samples. Important differences are that these samples include the short chain lipid, DCPC, such that the desired mole ratio (q) between the long chain lipid and short chain lipid is obtained, and the amount of buffer in the sample is significantly higher for bicelle samples than typically used for the multilamellar dispersions. DMPC-d54, DPoPC, and cholesterol were weighed out as dry powders, while the DCPC was added as an appropriate volume of a 2.5 mg/mL (DCPC/ethanol) stock solution due to its highly hygroscopic nature. The lipids were dissolved in ethanol, the solvent was removed by lyophilizing overnight, then the dry mixture was scraped from the round-bottomed flask. Buffer was added such that the final ratio of buffer weight/total hydrated sample weight (w/w) was 0.6. Bicelle samples were hydrated with 50 mM phosphate buffer except in cases where the bicelles were to be flipped to have their normals oriented parallel to the magnetic field with the use of lanthanide ions. In these cases, the buffer was mixed with lanthanide salts giving the final dry sample/lanthanide molar ratio of 10:1 when the weight ratio of buffer/total hydrated sample is maintained as 0.6. All samples were transferred into small glass tubes which were sealed with silicone to ensure that the water content was constant throughout the experiments. 2.3. Experimental setup 2 H NMR experiments were performed on Bruker BioSpin (Milton, ON) spectrometers at 76.77 MHz, 92.15 MHz, or 122.84 MHz using a quadrupolar echo pulse sequence [7]. Home-made coils were used and the 90° pulses were optimized and were kept as short as possible in order to minimize any artefacts [26]. The 90° pulse lengths used were 2.25 μs at 76.77 MHz, 1.70 μs at 92.15 MHz, and 2.75 μs at 122.84 MHz, the echo delay was 40 μs at 76.77 and 92.15 MHz, and 25 μs at 122.84 MHz. In the quadrupolar echo pulse sequence, 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 in the time domain and then the data points were shifted so that one point was precisely at the top of the echo. The points before the top of the echo were then removed. This is an important process which results in symmetric spectra with a flat baseline [27]. The temperature for each NMR probe was calibrated using Pb(NO3)2 [28,29], and the corrected temperatures are presented here. The melting points of DPPC-d62 and DMPC-d54 were used as references for the calibrations.

2.4. Moment analysis Moment analysis of deuterium spectra is a quantitative way to compare the molecular order of the phospholipids [30]. The nth moment of a spectrum can be defined as follows Mn ¼

1 A

Z

∞ −∞

  jω′jn f ω′ dω′

ð1Þ

where ω ′ = ω − ω0, ω0 is the Larmor frequency and f(ω′) is the function describing the lineshape of the spectrum. The area of the spectrum, A, is calculated as Z A¼

∞ −∞

  f ω′ dω′:

ð2Þ

The spectra are symmetric and the moments for the two halves of the spectra (from the negative frequency limit to the central frequency ω0 and from ω0 to the positive limit) are each calculated and the average is taken. The first moment is of particular interest here because it is directly proportional to the average carbon-deuterium bond order parameter,

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SCD. For a sample oriented with its bilayer normal perpendicular to the magnetic field, the bond order is related to the first moment as M1 ¼ 2π

 2  3e qQ hjSCD ji: 4h

ð3Þ

This type of moment calculation is developed in more detail in previous work [27]. In the present work, the first moments as a function of temperature are used to compare the molecular order and behaviour of bicelles with varying concentrations of cholesterol, as well as for bicelles aligned with their bilayer normals perpendicular and parallel to the magnetic field of the spectrometer. 3. Results 3.1. DMPC/DCPC/cholesterol bicelles A series of samples was made with different concentrations of cholesterol in DMPC-d54/DCPC bicelles (q = 3.2) with 60% buffer (buffer/ total hydrated sample (w/w)) to determine the effect of cholesterol on the phase behaviour of these bicelles. Samples will be referred to by the mole fraction of cholesterol (xc) in the sample, where xc = (mol cholesterol) / (mol DMPC + mol DCPC + mol cholesterol). 2 H NMR spectra of DMPC-d54/DCPC (q = 3.2) bicelles with no cholesterol (xc = 0) and with xc = 0.037 at temperatures from 316.8 K to 281.1 K are shown in Fig. 1. These samples are oriented bicelles over a broad temperature range, from about 293 K to over 320 K. An isotropic phase is observed at high temperatures (above those shown in the figure) and at lower temperatures, below 286.7 K in the absence of cholesterol and below 292.5 K with xc = 0.037. Fig. 2 shows the 2H spectra of DMPC-d54/DCPC (q = 3.2) bicelles with xc = 0.071 and xc = 0.10. Again note the isotropic phase that begins as the temperature is lowered. Two other compositions, with cholesterol mol fractions xc = 0.087 and xc = 0.12 (not shown), give results that are very similar to the xc = 0.10 bicelle sample but with the onset of the isotropic phase occurring at a temperature that is slightly lower for the xc = 0.087 sample, and slightly higher for the xc = 0.12 sample as compared to the xc = 0.10 case. Fig. 3 shows spectra for DMPC-d54/DCPC (q = 3.2) bicelles with xc = 0.13 and xc = 0.16. A major difference between these samples and all the other samples which had less cholesterol is the presence of a large isotropic peak throughout the entire temperature range investigated. The area of the isotropic part of the spectrum varies from 5% at higher temperatures to about 40% at lower temperatures. Because the isotropic phase is a significant fraction of the total sample under these conditions we cannot quantitate the compositions of the coexisting phases from the available data. The regions of the sample that are not in the isotropic phase appear to be well aligned within the magnetic field and give rise to relatively sharp peaks, many of which can be resolved from 304.5 K to 292.5 K in the xc = 0.13 sample and throughout the full range of temperatures shown for the xc = 0.16 sample. Although the spectra are not explicitly shown in Fig. 3(B), above 318 K two fluid ‘o − ‘d phase coexistence (like that seen above 304.5 K in Fig. 3(A)) is observed in the xc = 0.16 mol fraction cholesterol bicelles. At these levels of cholesterol, the quadrupolar splittings are much larger than those observed in the other bicelle samples, indicating that at high cholesterol content the predominant fluid phase is the more ordered ‘o phase. The phase behaviour of the DMPC-d54/DCPC bicelle mixtures (q = 3.2) with various concentrations of cholesterol is summarized in Fig. 4. The different phases and phase coexistences are represented by each of the different colours: the isotropic phase is magenta, the gel phase is green, the ‘d phase is violet, the gel + isotropic phase coexistence is yellow, the ‘o + isotropic phase coexistence is red, the ‘d + isotropic phase coexistence is cyan, and the ‘d + ‘o + isotropic phase coexistence is orange. As an example, consider the xc = 0.10 mol fraction cholesterol

Fig. 1. (A) DMPC-d54/DCPC bicelles (xc = 0), q = 3.2 at (a) 316.8 K, (b) 310.6 K, (c) 304.5 K, (d) 298.4 K, (e) 292.5 K, (f) 286.7 K, and (g) 281.1 K. Collected at 76.77 MHz, 512 scans. (B) DMPC-d54/DCPC/cholesterol bicelles, xc = 0.037, q = 3.2 at (a) 316.8 K, (b) 310.6 K, (c) 304.5 K, (d) 298.4 K, and (e) 292.5 K. Collected at 92.15 MHz, 512 scans.

bicelles which at the highest temperatures investigated here are magnetically aligned and in the ‘d phase down to about 302 K. Below that there is a rather gradual transition into a more ordered gel phase characterized by larger quadrupolar splittings and broader lines (with shorter T2). During this process the sample remains oriented although with splittings approaching those expected for the gel phase. In some of these spectra we see the inequivalence of the two chain methyls which is characteristic of phases with a high degree of chain order whether a result of a high cholesterol concentration or of being in the gel phase [4,31]. Eventually as the temperature is lowered further we find a gel phase (although for other concentrations we typically see a gel + isotropic two phase region) and finally the whole sample goes into an isotropic phase at low temperatures. The dashed line is used to represent the transition between the oriented ‘d phase and the gel phase. At higher temperatures for low cholesterol concentrations, an isotropic phase is observed. A persistent isotropic phase contribution to the spectra can

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Fig. 2. (A) DMPC-d54/DCPC/cholesterol bicelles, xc = 0.071, q = 3.2 at (a) 316.8 K, (b) 310.6 K, (c) 304.5 K, (d) 298.4 K, and (e) 292.5 K. Collected at 92.15 MHz, 512 scans. (B) DMPC-d54/DCPC/cholesterol bicelles, xc = 0.10, q = 3.2 at (a) 316.8 K, (b) 310.6 K, (c) 304.5 K, (d) 298.4 K, and (e) 292.5 K. Collected at 76.77 MHz, 512 scans.

also be seen in the xc = 0.13 and xc = 0.16 samples throughout the full range of temperatures. Data shown in the figure represents the full range of temperatures sampled in the experiments, from 341.5 K to 281.1 K. Note that not all temperatures were sampled for each cholesterol concentration. The upper limit for the oriented ‘d phase was not reached for the xc = 0.071 to xc = 0.12 bicelle samples. The first moments for the 2H spectra of the DMPC-d54/DCPC (q = 3.2) bicelles with varying concentrations of cholesterol were calculated as described above. The first moment gives a quantitative method for comparing the order and phase behaviour of the bicelles as a function of their cholesterol concentration and temperature. The calculations of the moments includes the entire spectrum for all samples except the xc = 0.13 and xc = 0.16 mol fraction cholesterol samples which have a large isotropic peak in the spectra. In the xc = 0.13 and xc = 0.16 samples the isotropic peak was excluded from the calculation of the

Fig. 3. (A) DMPC-d54/DCPC/cholesterol bicelles, xc = 0.13, q = 3.2 at (a) 316.8 K, (b) 310.6 K, (c) 304.5 K, (d) 298.4 K, and (e) 292.5 K. Collected at 92.15 MHz, 512 scans. (B) DMPC-d54/DCPC/cholesterol bicelles, xc = 0.16, q = 3.2 at (a) 316.8 K, (b) 310.6 K, (c) 304.5 K, (d) 298.4 K, and (e) 292.5 K. Collected at 92.15 MHz, 512 scans.

moments by cropping it out at the level at its base when the integration region for the moment calculation was selected. The isotropic peak in these spectra would affect the calculation of the first moment of the spectrum through its impact on the area because of its relative size. As a result, in order to determine the moments of the oriented part of the spectrum, we excluded the isotropic peak from the calculation. A comparison of the first moments over a temperature range from 330 K to 285 K for the entire series of samples from 0 to 0.16 mol fraction cholesterol in DMPC-d54/DCPC (q = 3.2) bicelles is shown in Fig. 5. In this figure the solid squares are for no cholesterol (xc = 0), the open circles are for xc = 0.037, the filled triangles are for xc = 0.071, the open down triangles are for xc = 0.087, the filled circles are for xc = 0.10, the open left triangles are for xc = 0.12, the filled right triangles are for xc = 0.13, and the open diamonds are for xc = 0.16. At high

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Fig. 4. Summary of phases observed in DMPC-d 54 /DCPC bicelle mixtures (q = 3.2) with varying concentrations of cholesterol. Each colour represents a different phase or coexisting phases: the isotropic phase is magenta, the gel phase is green, the ‘d phase is violet, the gel + isotropic phase coexistence is yellow, the ‘o + isotropic phase coexistence is red, the ‘d + isotropic phase coexistence is cyan, and the ‘d + ‘o + isotropic phase coexistence is orange. The dashed line indicates the transition between the oriented ‘d phase and the onset of the gel phases at lower temperatures. In the xc = 0.071 to xc = 0.12 bicelle samples the upper limit for the oriented ‘d phase was not reached.

temperatures, as the cholesterol concentration is raised the first moment increases. There is a large jump in the value of first moment of the NMR spectra between cholesterol concentrations of xc = 0.12 and xc = 0.13 due to the presence of the ‘o phase in the bicelles with xc = 0.13 and above. In addition, as the temperature is lowered, the first moment rises which indicates an increase in the amount of molecular order, or a decrease in the molecular mobility. There is a sharp drop in the first moment for the xc = 0.087, 0.10, and 0.12 samples near 300 K when the spectra become dominated by the isotropic phase. The temperature history for these types of samples is important and can affect the values of the calculated moments and the phase behaviour of the samples. These results show that there is a broad range of temperatures and compositions for which oriented bicelles containing DMPC, DCPC and cholesterol can be investigated. 3.2. DPoPC/DMPC/cholesterol/DCPC bicelles There has been a lot of work on ternary mixtures which exhibit two fluid phase coexistence using multilamellar dispersions or giant unilamellar vesicles (GUVs). Mixtures of DOPC, DPPC, and cholesterol have been studied by 2H NMR and phase diagrams for these mixtures have been published [2,6,32,33]. While it is possible to make bicelles with longer chained phospholipids, including DPPC, DMPC is used most frequently and easily produces well-oriented bicelles. In the current work, a ternary mixture analogous to DOPC, DPPC, cholesterol was used. DMPC has a chain length of 14 carbons while DPPC has a chain length of 16 carbons, consequently DPoPC (chain length: 16 carbons) was used instead of DOPC (chain length: 18 carbons) for the

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Fig. 5. Comparison of M1 for DMPC-d54/DCPC/cholesterol bicelles, q = 3.2 with varying concentrations of cholesterol over a range of temperatures from 285 K to 330 K. Solid squares: no cholesterol, open circles: xc = 0.037, filled triangle: xc = 0.071, open down triangle: xc = 0.087, filled circle: xc = 0.10, open left triangle: xc = 0.12, filled right triangle: xc = 0.13, and open diamond: xc = 0.16.

unsaturated lipid. Cholesterol was used in both cases. The phase behaviour, specifically the two phase coexistence of the ‘d and ‘o phases, for DPoPC/DMPC-d54/cholesterol multilamellar dispersions was established using 2H NMR. Fig. 6(A) shows the occurrence of the two phase ‘d–‘o region in the powder sample of 32:48:20 (DPoPC/DMPC-d54/cholesterol). This behaviour is similar to that found for DOPC/DPPC/cholesterol mixtures. Fig. 6(B) shows the occurrence of the ‘d–‘o phase coexistence in 32:48:20 (DPoPC/DMPC-d54/cholesterol) bicelles with q = 3.5 ((mol DPoPC + mol DMPC)/mol DCPC). A comparison between the 2H spectra of the powder and bicelle samples shows that a small fraction of the bicelle samples is in the ‘o phase even at high temperatures where the powder sample spectra show only the ‘d phase. The samples are hydrated and mixed at ambient temperature, so these mixtures are in the two phase region when they are prepared. As a result, some of the bicelles may be formed with a higher concentration of cholesterol than the sample average and consequently if these bicelles are not physically connected with the rest of the sample, they will not be able to exchange molecules with the bulk of the sample. Thus, there may be some isolated regions of the sample which even at high temperatures will exhibit the ‘o phase due to the high cholesterol content of those bicelles. The onset of the two phase region in these DPoPC/DMPC/cholesterol mixtures follows a trend similar to that observed in DOPC/DPPC/cholesterol mixtures, as the concentration of the saturated lipid (DMPC) is increased the two phase region occurs at a higher temperature. In addition, as shown in Fig. 7 the amount of ‘o phase increases as the composition of the bicelles changes from 32:48:20 (DPoPC/DMPC-d54/ cholesterol) to 30:55:15 (DPoPC/DMPC-d54/cholesterol) to 20:60:20 (DPoPC/DMPC-d54/cholesterol) with q = 3.5 ((mol DPoPC + mol DMPC)/mol DCPC) at 299.5 K. Finally, we should mention that we have found that mixtures of DOPC/DPPC/cholesterol with DCPC also form oriented bicelles which can exhibit ‘d–‘o phase coexistence (data not shown).

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Fig. 7. Bicelles with q = 3.5 ((mol DPoPC + mol DMPC)/mol DCPC) (a) 32:48:20 (DPoPC/ DMPC-d54/cholesterol), (b) 30:55:15 (DPoPC/DMPC-d54/cholesterol), and (c) 20:60:20 (DPoPC/DMPC-d54/cholesterol) at 299.5 K. (a) collected at 92.15 MHz, (b) collected at 122.84 MHz, and (c) collected at 76.77 MHz, all have 1024 scans.

peak of the bicelles with the plain buffer and serves as a visual guide for comparing the three spectra. Two phase coexistence between the ‘d and ‘o phases is observed in the flipped bicelles and spectra showing the onset of this two phase region in 32:48:20 (DPoPC/DMPC-d54/cholesterol) + DCPC bicelles, q = 3.5 are shown in Fig. 9(A). Since the quadrupolar splittings of the bicelles with their normal oriented parallel to the magnetic field (flipped by Yb3+) should be twice those of the bicelles oriented perpendicular to the magnetic field, the first moment for the flipped bicelles should be twice that of the ordinary bicelles. Indeed, this can be seen in Fig. 9(B) which shows both sets of moments (squares are for the sample with plain buffer (no lanthanide), and the triangles are for the buffer with Yb3+) and also 2 × M1 (circles) for the bicelles made with plain buffer. At high temperatures and again at low temperatures M1 with YbCl3 and 2 × M1 with plain buffer are in good agreement. However,

Fig. 6. The onset of the two phase ℓd–ℓo region in powder (A) and bicelle (B) samples. (A) 32:48:20 (DPoPC/DMPC-d54/cholesterol) at (a) 294.8 K, (b) 293.7 K, (c) 292.5 K, (d) 291.3 K, and (e) 290.2 K. Collected at 92.15 MHz, 1024 scans. (B) 32:48:20 (DPoPC/ DMPC-d54/cholesterol) bicelles, q = 3.5 ((mol DPoPC + mol DMPC)/mol DCPC) at (a) 306.9 K, (b) 300.8 K, (c) 294.8 K, (d) 289.0 K, and (e) 283.3 K. Collected at 92.15 MHz, 1024 scans.

3.3. Bicelles and lanthanide ions Lanthanide ions can be used to flip the orientation of bicelles in a magnetic field, which may be advantageous for samples containing proteins or peptides [34,35]. Two different lanthanide salts, EuCl 3 and YbCl 3, were added to the stock 50 mM phosphate buffer in order to determine which performed better for the DPoPC/DMPCd 54 /cholesterol + DCPC bicelles. The weight ratio of buffer/total hydrated sample in these samples was always 0.6, and the final dry sample/lanthanide molar ratio was 10:1. Fig. 8 demonstrates the effect of adding the two different lanthanide salts to the bicelle mixtures and compares these spectra to a sample made without any lanthanide salts. The dashed vertical line indicates the outer

Fig. 8. The effect of adding two different lanthanides to 32:48:20 (DPoPC/DMPC-d54/ cholesterol) with DCPC, q = 3.5 bicelles (dry sample/lanthanide molar ratio 10:1). (a) 50 mM phosphate buffer (no lanthanide), (b) 50 mM phosphate buffer with EuCl3, and (c) 50 mM phosphate buffer with YbCl3. Spectrometer 2H frequency: 92.15 MHz, 1024 scans, 316.8 K.

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Fig. 9. (A) Onset of the two phase region in flipped 32:48:20 (DPoPC/DMPC-d54/ cholesterol) + DCPC bicelles, q = 3.5 with YbCl3 in the 50 mM phosphate buffer at (a) 304.5 K, (b) 298.4 K, (c) 292.5 K, (d) 286.7 K, and (e) 281.1 K. (B) Comparison of the first moment of the 32:48:20 (DPoPC/DMPC-d54/cholesterol) + DCPC q = 3.5 bicelle spectra with plain buffer (M1:squares and 2 × M1:circles) and buffer with YbCl3 (triangles).

the shape of the curves of the first moment as a function of temperature differs between the sample with and without Yb3+. This indicates that presence of the lanthanide ions has an effect on the phase behaviour of the bicelles. 4. Conclusions For DMPC-d54/DCPC (q = 3.2) bicelles with cholesterol concentrations varying from xc = 0 to 0.16, M1 of the 2H spectra indicates that at high temperatures M1 increases as the relative amount of cholesterol increases. This result is expected since cholesterol is known to have a stiffening effect on the phospholipid chains and allows for the formation of a more ordered fluid phase (‘o). At high temperature there is a significant change in the values of M1 between the 0.12 and 0.13 mol fraction cholesterol bicelles due to the presence of the liquid ordered phase at the higher cholesterol concentration. As the temperature is lowered, the first moment rises which again indicates an increase in the amount of molecular order. At lower temperatures, there is a conversion to an

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isotropic phase for all samples. The isotropic phase is also present in samples with high cholesterol concentrations (0.13 and 0.16 mol fraction cholesterol) over the entire temperature range investigated. Unfortunately, the presence of the isotropic phase makes it difficult to determine the composition of the coexisting phases under these conditions. Significantly, we observe two fluid phase coexistence in these bicelles even though there is no unsaturated phospholipid present. There is a relatively wide range of temperatures and compositions of the DMPC/cholesterol/DCPC bicelles which align in the magnetic field and can provide an environment for investigating peptides or proteins incorporated into a membrane. Two fluid phase coexistence has been observed in multilamellar dispersions containing one unsaturated phospholipid and one saturated phospholipid (DOPC + DPPC or DPoPC + DMPC) along with cholesterol. Bicelles can be formed by combining these DPoPC/DMPC/cholesterol mixtures with the short chain lipid DCPC and coexisting ‘d and ‘o fluid phases were observed over large temperature and composition ranges. These coexisting oriented phases occur with little or no isotropic phase being present. Bicelles which exhibit coexisting ‘d and ‘o fluid phase domains could provide a medium in which to study membrane proteins and peptides under different phase conditions by varying the temperature and composition. Just as for multilamellar dispersions, varying the composition of DPoPC/DMPC-d54/cholesterol in the bicelles gives rise to increasing amounts of the ‘o phase as either the concentration of DMPC and/or of cholesterol increases. We find that even at high temperatures, a small amount of ‘o phase is observed in the 2H spectra. This contribution to the spectra likely comes from a small fraction of the bicelles which contain a higher concentration of cholesterol. If some of the bicelles are not physically connected and not free to exchange molecules with the rest of the sample, they can display different phase behaviour than the bulk sample and this is a challenge for studying the partitioning of peptides or proteins into the different fluid phases. One possible solution may be to prepare multicomponent bicelle samples at a higher temperature where a single fluid phase is expected and the distribution of cholesterol will be more homogeneous. Nonetheless, if there exists a significant fraction of bicelles which are isolated from the rest of the sample, i.e., are unable to exchange molecules with other bicelles, then the interpretation of such studies may be more difficult. It is well established that lanthanide ions can be used to flip bicelles. DMPC/DCPC bicelles typically align such that their bilayer normals are perpendicular to the external magnetic field. In the presence of lanthanide ions, these bicelles align such that their bilayer normals are parallel with the external magnetic field. Bicelles containing DPoPC/DMPC/ cholesterol and DCPC were found to exhibit ‘d–‘o phase coexistence in the parallel orientation when YbCl3 was added to the buffer. It was found that the addition of EuCl3 did not flip the bicelles when cholesterol was present and it interfered with the alignment/structure of the bicelles. This ‘flipped’ orientation can be advantageous when peptides/ proteins are present whose diamagnetic susceptibility would promote a parallel alignment with the magnetic field. In addition, this orientation is preferable when studying peptides or proteins in oriented samples since the spectra can be used to determine directly the orientation of the relevant bonds with respect to the external magnetic field (which is aligned along the bilayer normal) even in the absence of axial reorientation. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (Grant Number RG2995), the

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Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation. M.L. Schmidt was the recipient of an Alexander Graham Bell Canada Graduate Scholarship (2011–2014) and an Ontario Graduate Scholarship (2014–2015). We would like to thank the staff of the University of Guelph NMR Centre for their help with the instrumentation. References [1] M.R. Vist, J.H. Davis, Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry, Biochemistry 29 (1990) 451–464, http://dx.doi.org/10.1021/bi00454a021. [2] J.H. Davis, J.J. Clair, J. Juhasz, Phase equilibria in DOPC/DPPC-d62/cholesterol mixtures, Biophys. J. 96 (2) (2009) 521–539, http://dx.doi.org/10.1016/j.bpj.2008.09. 042. [3] J.H. Davis, L. Ziani, M.L. Schmidt, Critical fluctuations in DOPC/DPPC-d62/cholesterol mixtures: 2H magnetic resonance and relaxation, J. Chem. Phys. 139 (2013) 045104, http://dx.doi.org/10.1063/1.4816366. [4] J.H. Davis, M.L. Schmidt, Critical behaviour in DOPC/DPPC/cholesterol mixtures: static 2H NMR line shapes near the critical point, Biophys. J. 106 (2014) 1970–1978, http://dx.doi.org/10.1016/j.bpj.2014.03.037. [5] S.L. Veatch, I.V. Polozov, K. Gawrisch, S.L. Keller, Liquid domains in vesicles investigated by NMR and fluorescence microscopy, Biophys. J. 86 (5) (2004) 2910–2922, http://dx.doi.org/10.1016/S0006-3495(04)74342-8. [6] S.L. Veatch, O. Soubias, S.L. Keller, K. Gawrisch, Critical fluctuations in domainforming lipid mixtures, Proc. Natl. Acad. Sci. 104 (2007) 17650–17655, http://dx. doi.org/10.1073/pnas.0703513104. [7] J.H. Davis, K.R. Jeffrey, M. Bloom, M.I. Valic, Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains, Chem. Phys. Lett. 42 (2) (1976) 390–394, http://dx.doi.org/10.1016/0009-2614(76)80392-2. [8] C.R. Sanders, J.P. Schwonek, Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR, Biochemistry 31 (37) (1992) 8898–8905, http://dx.doi.org/10. 1021/bi00152a029. [9] R.R. Vold, R.S. Prosser, Magnetically oriented phospholipid bilayered micelles for structural studies of polypeptides. Does the ideal bicelle exist? J. Magn. Reson. B 113 (1996) 267–271, http://dx.doi.org/10.1006/jmrb.1996.0187. [10] C.R. Sanders, R.S. Prosser, Bicelles: a model membrane system for all seasons? Structure 6 (10) (1998) 1227–1234, http://dx.doi.org/10.1016/S0969-2126(98)00123-3. [11] I. Marcotte, M. Auger, Bicelles as model membranes for solid-and solution-state NMR studies of membrane peptides and proteins, Concepts Magn. Reson. Part A 24 (1) (2005) 17–37, http://dx.doi.org/10.1002/cmr.a.20025. [12] U.H.N. Dürr, M. Gildenberg, A. Ramamoorthy, The magic of bicelles lights up membrane protein structure, Chem. Rev. 112 (2012) 6054–6074, http://dx.doi.org/10. 1021/cr300061w. [13] U.H.N. Dürr, R. Soong, A. Ramamoorthy, When detergent meets bilayer: birth and coming of age of lipid bicelles, Prog. Nucl. Magn. Reson. Spectrosc. 69 (2013) 1–22, http://dx.doi.org/10.1016/j.pnmrs.2013.01.001. [14] D.E. Warschawski, A.A. Arnold, M. Beaugrand, A. Gravel, E. Chartrand, I. Marcotte, Choosing membrane mimetics for NMR structural studies of transmembrane proteins, Biochim. Biophys. Acta 1801 (2011) 1957–1974, http://dx.doi.org/10.1016/j. bbamem.2011.03.016. [15] R.S. Prosser, F. Evanics, J.L. Kitevski, M.S. Al-Abdul-Wahid, Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins, Biochemistry 45 (28) (2006) 8453, http://dx.doi.org/10.1021/bi060615u. [16] A. Diller, C. Loudet, F. Aussenac, G. Raffard, S. Fournier, M. Laguerre, A. Grelard, S.J. Opella, F.M. Marassi, E.J. Dufourc, Bicelles: a natural ‘molecular goniometer’ for structural, dynamical and topological studies of molecules in membranes, Biochimie 91 (2009) 744–751, http://dx.doi.org/10.1016/j.biochi.2009.02.003. [17] M. Liebi, J. Kohlbrecher, T. Ishikawa, P. Fischer, P. Walde, E.J. Windhab, Cholesterol increases the magnetic aligning of bicellar disks from an aqueous mixture of

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