DHPC Aggregates

The morphology of DMPC/DHPC mixtures at total lipid concentration cL ) 5% (w/w) and DMPC/DHPC ratio q ≈ 3, doped with small amounts of DMPG or CTAB,...
1 downloads 0 Views 290KB Size
Download PDF
3280

Langmuir 2006, 22, 3280-3285

Morphology of Magnetically Aligning DMPC/DHPC AggregatessPerforated Sheets, Not Disks Lorens van Dam, Go¨ran Karlsson, and Katarina Edwards* Department of Physical and Analytical Chemistry, Uppsala UniVersity, Box 579, SE-751 23 Uppsala, Sweden ReceiVed NoVember 7, 2005. In Final Form: January 12, 2006 The morphology of DMPC/DHPC mixtures at total lipid concentration cL ) 5% (w/w) and DMPC/DHPC ratio q ≈ 3, doped with small amounts of DMPG or CTAB, was investigated. 31P NMR was used to identify the magnetically aligning phase, and cryo-transmission electron microscopy (cryo-TEM) was employed for structural characterization. Magnetic alignment was found to occur between ∼30 and ∼45 °C, and cryo-TEM showed that the magnetically aligning phase consisted of extended sheets with a lacelike structure. The aggregates are best described as intermediates between two-dimensional networks of flattened, highly branched, cylindrical micelles and lamellar sheets perforated by large irregular holes. DHPC most likely covers the edges of the holes, while DMPC makes up the bilayer bulk of the aggregates. However, 20-43% of the DHPC takes part in the bilayer, corresponding to 6-12% of the bilayer being made up of DHPC. This fraction increases with increasing temperature. At temperatures above 45 °C, the aligning phase collapses.

Introduction Considerable interest has been focused on the aggregates formed in mixtures of the short-chain phospholipid DHPC (dihexanoylphosphatidylcholine, di-C6 lecithin) and the longchain phospholipid DMPC (dimyristoylphosphatidylcholine, diC14 lecithin). From the NMR point of view, there are two reasons for this attention. First, aqueous mixtures at low DMPC:DHPC ratios, typically 1:2, form small (nonaligning) disks that may be employed as membrane mimics in high-resolution NMR of membrane-associated proteins and polypeptides.1,2 Second, the aggregates formed at higher DMPC:DHPC ratios, typically 3:1, align in magnetic fields and may be used to yield a small anisotropy in the rotational motion of nonspherical water-soluble proteins.3,4 The resulting residual dipolar couplings from highresolution NMR spectra aid in the structural characterization of the dissolved biomolecules.5-7 Aligned DMPC/DHPC phases may, furthermore, be employed in solid-state NMR investigations of membrane-associated proteins.8 The present investigation focuses on the magnetically aligning phase found under the conditions employed in high-resolution NMR investigations of water-soluble biomolecules. Initially, the magnetically aligning aggregates used in highresolution (liquid state) NMR were believed to have a discoidal shape, just like the aggregates formed at lower DMPC:DHPC ratios.8 The disks are frequently termed “bicelles” in the NMR community and are ideally modeled as consisting of a circular bilayer of DMPC enclosed by a rim of DHPC.3 Recent experimental results indicate, however, that the magnetically aligning aggregates are not disk-shaped.9-14 Instead, extended * Corresponding author. E-mail: [email protected]. (1) Vold, R. R.; Prosser, R. S.; Deese, A. J. J. Biomol. NMR 1997, 9, 329-335. (2) Sanders, C. R., II.; Oxenoid, K. Biochim. Biophys. Acta 2000, 1508, 129145. (3) Vold, R. R.; Prosser, R. S. J. Magn. Reson. B 1996, 113, 267-271. (4) Ottiger, M.; Bax, A. J. Biomol. NMR 1998, 12, 361-372. (5) Tjandra, N.; Bax, A. Science 1997, 278, 1111-1114. (6) Prestegard, J. H.; Kishore, A. I. Curr. Opin. Chem. Biol. 2001, 5, 584-590. (7) Bax, A. Protein Sci. 2003, 12, 1-16. (8) Sanders, C. R., II; Hare, B. J.; Howard, K. P.; Prestegard, J. H. Prog. NMR Spectrosc. 1994, 26, 421-444. (9) Gaemers, S.; Bax, A. J. Am. Chem. Soc. 2001, 123, 12343-12352. (10) Nieh, M.-P.; Glinka, C. J.; Krueger, S. Langmuir 2001, 17, 2629-2638.

lamellar sheets of DMPC, perforated by DHPC-lined holes, have been suggested as a more likely structure.9,10,13,15 A problem with DMPC/DHPC mixtures is that a macroscopic phase separation frequently occurs as the temperature is increased to the region where the aligning phase is found (typically 30-40 °C, depending on the DMPC/DHPC ratio).4,16 An aligning phase that does not phase-separate can be achieved, however, by temperature jumping from below the DMPC main transition temperature, Tm ) 24 °C.4 Alternatively, the system may be stabilized by inclusion of small amounts of charged amphiphiles.16 In this study, we investigate the morphology of magnetically aligning DMPC/DHPC aggregates doped with small amounts of the charged amphiphiles dimyristoylphosphatidylglycerol (DMPG) or N-cetyl-N,N,N-trimethylammonium bromide (CTAB). 31P NMR and cryo-TEM are used in combination. The former reports on the magnetic alignability of the phase and on the local surroundings of the amphiphiles, while the latter provides details on the sample morphology via direct visualization of the aggregates. The study extends our previous investigation of the pure DMPC/DHPC system, wherein the phase diagram and the aggregate structures at different compositions and temperatures were investigated.13 Materials and Methods Chemicals and Sample Preparation. Dihexanoylphosphatidylcholine (DHPC), dimyristoylphosphatidylcholine (DMPC), and the sodium salt of DMPG were purchased as dry powders form Avanti Polar Lipids (Alabaster, AL) and used as received. Analythical grade CTAB was purchased in powdered form from Merck (Darmstadt, Germany) and used as received. Separate stock solutions were prepared by weighing in appropriate amounts of either chemical and buffer. Stock concentrations were (11) Nieh, M.-P., Glinka, C. J.; Krueger, S.; Prosser, R. S.; Katsaras, J. Biophys. J. 2002, 82, 2487-2498. (12) Rowe, B. A.; Neal, S. L. Langmuir 2003, 19, 2039-2048. (13) van Dam, L.; Karlsson, G.; Edwards, K. Biochim. Biophys. Acta 2004, 1664, 241-256. (14) Nieh, M.-P.; Raghunathan, V. A.; Glinka, C. J.; Harroun, T. A.; G. Pabst, G.; Katsaras, J. Langmuir 2004, 20, 7893-7897. (15) Soong, R.; Macdonald, P. M. Biophys. J. 2005, 88, 255-268. (16) Losonczi, J. A.; Prestegard, J. H. J. Biomol. NMR 1998, 12, 447-451.

10.1021/la052988m CCC: $33.50 © 2006 American Chemical Society Published on Web 02/22/2006

Magnetically Aligning DMPC/DHPC Aggregates

Langmuir, Vol. 22, No. 7, 2006 3281

100 mM for DHPC and DMPC and 20 mM for DMPG and CTAB. DHPC and CTAB readily dissolve in water. The water-insoluble DMPC and DMPG were heated to about 45 °C and vigorously vortexed, which produced homogeneous stock dispersions. We used a 10 mM sodiumphosphate buffer without additional salt, prepared at pH 6.5. At this pH the rate of phospholipid hydrolysis is at a minimum.17 The buffer also contained 0.01% NaN3 in order to avoid bacterial growth. Samples were prepared by mixing appropriate amounts (by volume) of chemicals, taken from the stock solutions, buffer and D2O (>99%). Appropriate amounts were calculated from the following relations: m(DMPC) + m(DHPC) + m(X) cL ) m(solvent) qX )

[DMPC] + [X] [DHPC]

[X] f) [DMPC] + [X]

(1)

[DMPC] + [DMPG] [DHPC]

sample

Tfv

Tct

∆Tv

no additive f ) 2% DMPG f ) 2% CTAB

24 24 25

27 28 30

4 4 6

a Tfv is the transition temperature from a fluid to a viscous phase, Tct is the transition temperature from a clear to a turbid phase, and ∆Tv is the temperature span of the viscous phase.

is blotted away, and the grid is then immediately plunged into liquid ethane at -165 °C. (4). The grid is mounted into the microscopy specimen holder and then transferred in a liquid nitrogen cooled transfer unit to the microscope.

(2)

Results (3)

with X denoting DMPG or CTAB. All samples were prepared at cL ) 5% and qX ) 3.2. f ) 2% was used in the DMPG/CTAB-containing samples. The samples contained 10% D2O (by volume) for NMR field locking, so that the sample buffer (phosphate) concentration was 9 mM. The samples were heated to 45 °C, vigorously vortexed, cooled to 10 °C, and vortexed again, a cycle that was repeated three or more times (until no change in sample appearance could be observed upon further cycling). Samples were stored in a freezer between measurements and were always subjected to several heating/ cooling cycles (45 °C/10 °C) with vortexing at both temperature extremes before use. As we shall use 31P to characterize the samples, it is convenient to define a ratio, qP, between the phosphorus-containing amphiphilic sample constituents (all except CTAB) as qP )

Table 1. Macroscopically Observable Phase-Transition Temperatures of the DMPC/DHPC System with and without Charge-Dopinga

(4)

For the DMPG-doped sample, qP ) qX ) 3.2, while for the CTABdoped sample, qX ) 3.2 and qP ) 3.14. NMR. 31P NMR was performed by employing a Varian Mercury 300 system equipped with a 3.5 T superconducting magnet, yielding a 1H Larmor frequency of 300 MHz and a 31P Larmor frequency of 121 MHz. The experiment consisted of a single 90° 31P pulse (19 µs duration) followed by acquisition under 2.5 kHz 1H WALTZ decoupling. The acquisition time was 256 ms, during which 4096 complex points were recorded. The spectral width was 8000 Hz, and 128 acquisitions were added for each spectrum, with a delay of 5 s between successive experiments. This delay was enough for complete relaxation of all 31P NMR resonances. FID’s were Fourier transformed using 5 Hz exponential line-broadening and phased to first-order, to produce 31P NMR spectra in absorption mode. The phosphate buffer 31P resonance was used as internal chemical shift reference at 0 ppm in all spectra. Cryo-TEM. Electron microscopy was performed using a Zeiss EM 902A transmission electron microscope (LEO Electron Microscopy, Oberkochen, Germany) operating at 80 kV in zero-loss bright-field mode. Digital images were recorded using a BioVision Pro-SM Slow Scan CCD camera (Proscan GmbH, Scheuring, Germany), giving a digital resolution corresponding to a pixel size of 1.6 × 1.6 nm at the magnification used (105×). The sample preparation procedure has recently been reviewed18 and consists of the following steps: (1) The solution to be investigated is left in a thermostated chamber at (close to) 100% relative humidity. (2) Typically, 1 µL of the solution is taken out with a pipet and deposited on a Cu grid covered by a holey polymer film. (3) Excess solution (17) Grit, M.; Crommelin, D. J. A. Chem. Phys. Lipids 1993, 64, 3-18. (18) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf. A 2000, 174, 3-21.

Ocular Observations. The DMPG- and CTAB-doped samples showed with increasing temperature the same succession of macroscopic phase transitions. Below the DMPC main transition temperature at 24 °C, the samples appeared optically clear and displayed a fluidity approximately that of water. As the DMPC main transition temperature was passed, the samples turned viscous and, upon further elevation of the temperature, gradually became more turbid. With increasing turbidity the viscosity leveled off. The turbidity of the pure (i.e., uncharged) DMPC/ DHPC mixture was much higher than that of the DMPG- and CTAB-containing (i.e., charged) samples. The two latter samples showed only a vague, slightly bluish, turbidity, and the samples were translucent. The former (uncharged) sample was milky white and completely nontransparent. Macroscopic phase separation eventually occurred in the pure DMPC/DHPC sample at temperatures above approximately 30 °C, resulting in a white, very turbid phase at the bottom and a transparent solution on top in the sample vial. The macroscopic phase-transition temperatures, as observed by the unaided eye when the samples were slowly heated, are collected in Table 1. In this table, Tfv is the temperature where the sample starts to become viscous (the fluid-viscous transition temperature), while Tct is the temperature where the sample becomes turbid (the clear-turbid transition temperature). The data displayed in Table 1 show that inclusion of DMPG does not affect Tfv and Tct. Inclusion of CTAB, however, has the effect of slightly increasing both transition temperatures. 31P NMR Spectra. Figure 1 shows the 31P NMR spectra of the DMPG- and CTAB-doped samples as a function of temperature. The spectra are similar to those observed by others under similar conditions.4,9,19 Below the DMPC main transition temperature (24 °C), both samples show (apart from the buffer resonance at 0 ppm) a single resonance at -1.4 ppm (bottom row in Figure 1). For the DMPGdoped sample, inspection of the baseline reveals some broadly distributed intensity. The CTAB-containing sample, in contrast, shows a seemingly unperturbed baseline. At 25 °C, i.e., just above the DMPC main transition temperature, the CTAB-containing sample still shows a single resonance at -1.4 ppm. The spectrum of the DMPG-containing sample, however, has changed markedly compared to the situation at 20 °C. The resonance at -1.4 ppm has substantially diminished, and a new, very broad resonance centered at around -10 ppm has appeared. (19) Triba, M. N.; Warschawski, D. E.; Deveaux, P. F. Biophys. J. 2005, 88, 1887-1901.

3282 Langmuir, Vol. 22, No. 7, 2006

Van Dam et al.

Figure 1. 31P NMR spectra under 1H WALTZ decoupling as a function of temperature of the DMPG-doped DMPC/DHPC mixture (left column) and the CTAB-doped DMPC/DHPC mixture (right column). The temperature is indicated in the figure and was increased smoothly starting from ∼10 °C; 5 Hz exponential line-broadening was used. The vertical scaling is the same in all spectra, except for the spectra of the lower row (T ) 20 °C), which have been scaled down by a factor 0.8 compared to the other spectra.

At 30 °C, both the CTAB- and DMPG-doped samples show similar spectral features, with two distinct resonances (apart from the buffer resonance at 0 ppm). The spectra are very different from those at the two lower temperatures. For the DMPGcontaining sample, the resonances are situated at -3.7 and -12.0 ppm, while for the CTAB-containing sample, the spectral lines appear at -3.4 and -11.5 ppm. The sample containing CTAB shows a small resonance also at -1.4 ppm, i.e., at the position where all spectral intensity was located at 25 °C. Throughout the temperature range 30-45 °C, the overall spectral appearance does not change for either of the doped samples. However, the two observed resonances move upfield (toward the right) with increasing temperature. For the DMPGcontaining sample, the shifts are around -0.2 ppm/°C for the downfield (leftmost) peak and -0.08 ppm/°C for the upfield (rightmost) peak. The CTAB-containing sample shows a shift of around -0.16 ppm/°C for the downfield peak and -0.08 ppm/°C for the upfield peak. At 45 °C, the resonances are located at -6.5 and -13.3 ppm for the DMPG-doped sample, and at -5.7 and -12.9 ppm for the CTAB-doped sample, respectively. The relative intensities (integrated) of the two peaks is 3.0 ( 0.1 for both samples throughout the temperature range 30-45 °C, corresponding well to their values of qP (see eq 4 for definition). At 50 °C, the 31P spectra change markedly for both doped samples, as seen in the top row of Figure 1. The two resonances

Figure 2. Cryo-TEM micrographs of the DMPG-doped sample at 20 °C (a), 25 °C (b), and 30 °C (c). Arrows marked A and B in part a denote disks viewed edge-on and face-on, respectively. Arrows C and D in part b denote branched and toroidal structures, respectively. Note the overlapping sheet at the top right of part c. Bar ) 100 nm.

seen at 45 °C and below have diminished and broadened, while a new peak arises at -1.4 ppm. The chemical shift value of this peak is strikingly similar to that of the peak seen at the two lowest temperatures (20 and 25 °C). At 60 °C, the spectra are similar to those at 50 °C, with some more intensity shifted toward the peak at -1.4 ppm (data not shown). Upon cooling from 60 to 40 °C, the spectra look the same as when heated from 20 °C, showing that the transitions are reversible. This indicates, in turn, that the spectra shown in Figure 1 correspond to samples in thermodynamic equilibrium (rather than to kinetically trapped samples). Cryo-TEM Observations. Figure 2 shows cryo-TEM images of the DMPG-doped sample used in the NMR experiments at various temperatures. The CTAB-doped sample displayed virtually identical morphologies, albeit at slightly shifted temperatures (see Table 1 and Figure 1), and therefore no images are shown.

Magnetically Aligning DMPC/DHPC Aggregates

At 20 °C, i.e., below the main transition temperature of DMPC, discoidal aggregates are seen to dominate the sample morphology (Figure 2a). The disks are most easily observed when oriented with their normal perpendicular to the direction of the electron beam (“edge-on”, arrow A). A disk seen “face-on”, i.e., with its normal parallel to the electron beam, has also been marked out (arrow B). At 25 °C (Figure 2b), the DMPG-doped sample still contains a fraction of discoidal aggregates. In addition, long threadlike cylindrical micelles are frequently observed. The latter show a strong tendency to branch (arrow C) and loop (arrow D). The cylindrical micelles appear to be somewhat flattened, a feature that was also seen in a previous study from this laboratory concerning the pure (i.e. nondoped) DMPC/DHPC system.13 At 30 °C (Figure 2c), the DMPG-doped sample has changed character and is now dominated by extended aggregates exhibiting an interesting, somewhat lacelike morphology. The structure may be described as intermediate between a two-dimensional network of branched, or interconnected, flattened cylindrical micelles and an extended lamellar sheet perforated by large irregular holes. Essentially the same structure is observed at 40 °C, although the network now appears more condensed (results not shown). It should be noted that the cryo-TEM preparation technique excludes very large (>µm) aggregates due to the limited thickness of the vitrified film.18 Further, at the high concentrations used in the present study (cL ) 5%) aggregates situated at different depths in the film frequently overlap in the images and may thus confuse the interpretation of the structure. In order avoid this problem and to better reveal the structural elements, the micrographs displayed in Figure 2 were chosen from less dense parts of the samples.

Discussion Effects of DMPG and CTAB on General Phase Behavior. We recently investigated the sample morphology in the pure (i.e., undoped) DMPC/DHPC system as a function of total phospholipid concentration (cL), DMPC/DHPC ratio (q), and temperature. It was found that, as the ratio q or the temperature is increased, the system goes through a general sequence of structural transitions. The initially discoidal micelles, found at low q and/or low temperature, transform into long somewhat flattened cylindrical micelles, which branch into networks and finally condense into perforated lamellar sheets.13 At q-values around 3, the cylindrical micelles appear just above the DMPC main transition temperature (24 °C). The presence of the long, threadlike, micelles is macroscopically manifested as a marked increase in sample viscosity. Upon formation of the extended networks, the viscosity then drops again and concomitantly the sample begins to show turbidity. The macroscopically observable phase-transitions of the DMPG- and CTAB-doped samples, i.e., the onset of viscosity and turbidity as temperature is elevated, are similar to those observed for the pure DMPC/DHPC-system. From Table 1 it is seen that the transition temperatures Tfv, the temperature where the sample viscosity starts to increase, and Tct, the temperature where sample turbidity sets in, are not affected by the addition of DMPG. Interestingly, the addition of CTAB seems to increase the transition temperatures somewhat. In our previous study of the pure DMPC/DHPC it was seen that a decrease in the DMPC/ DHPC ratio q has the effect of increasing Tfv and Tct, as well as to increase the temperature span over which the sample is viscous, ∆Tv.13 It thus appears that the addition of CTAB has a similar effect as decreasing the q-value (i.e., increasing the relative amount

Langmuir, Vol. 22, No. 7, 2006 3283

of DHPC in a pure DMPC/DHPC system). This may be explained by the fact that both CTAB and DHPC are micelle-forming surfactants and thus prefer to aggregate into structures of high positive curvature (like a micelle or the rim of a disk). Substitution of DMPC for DMPG, however, appears to have no effect on the transition temperatures Tfv and Tct, which may be expected due to their structural similarity and therefore similar preference to an environment of (close to) zero curvature. Structure and Spectroscopic Behavior at Low Temperatures. The cryo-TEM images displayed in Figure 2 show that the structures formed in the DMPG-doped sample are similar to those found in the pure DMPC/DHPC system at the corresponding temperatures. At 20 °C, i.e., below the DMPC main transition temperature, discoidal micelles dominate the sample (Figure 2a). The 31P NMR spectra recorded at this temperature (Figure 1) essentially consist of a single isotropic line at -1.4 ppm. Close inspection reveals that there are actually two peaks with slightly different chemical shifts, which could reflect the slightly differing environments of the bilayer and the rim (see ref 19 for a further discussion on this possibility). Pure DHPC solutions at 50 mM yield a single narrow 31P line at a chemical shift of -1.36 ppm (data not shown). However, at a DHPC concentration of 10 mM, the chemical shift reads -1.22 ppm (data not shown). The difference in chemical shift may be traced to the formation of micelles, the critical micellar concentration of DHPC being 14-15 mM.13 The chemical shift of -1.36 ppm in 50 mM DHPC is therefore a population-weighted average between DHPC in the micellar and in the monomeric forms (assuming rapid exchange occurs), which leads, using the standard two-site rapid-exchange model, to an expected DHPC micellar chemical shift of -1.42 ppm. The 31P NMR spectra of the doped DMPC/DHPC mixtures show at 20 °C (bottom row of Figure 1) a chemical shift value close to that expected for micellar DHPC, indicating that small micellar-like aggregates are present. This is in line with the cryoTEM observation of discoidal aggregates at this temperature (Figure 2a). The phosphatidylcholine headgroups are identical for DHPC and DMPC, and thus, the small (rapidly tumbling) disks may well display a chemical shift value similar to that of pure DHPC micelles. In our previous study,13 we also found discoidal micelles to be dominating below the DMPC main transition temperature. At the high q-value of 6, the disks coexisted with bilayer sheets of presumably pure DMPC, and the disk size did not increase beyond 20 nm diameter, corresponding, by virtue of eq 4, to q ) qP ) 2.5. The broad intensity that appears almost buried in the baseline of the 31P spectrum of the DMPG-doped sample at 20 °C could arise from bilayer sheets such as those observed in our previous study at q ) 6. At 25 °C the CTAB-doped sample shows a 31P spectrum similar to that at 20 °C, although the distorted baseline indicates a broad resonance similar to that seen for the DMPG-doped sample at 20 °C. The DMPG-doped sample displays at 25 °C, however, an additional broad resonance at -10 ppm. Further, the spectral line at -1.4 ppm is significantly diminished, as well as broadened, by the temperature increase from 20 to 25 °C. The cryo-TEM image displayed in Figure 2b, reveals that the DMPG-doped sample at 25 °C contains long, and frequently interconnected, cylindrical micelles in coexistence with the discoidal aggregates. As previously reported for the pure DMPC/DHPC system,13 the cylindrical micelles appear to be slightly flattened. The broad resonance at -10 ppm in the 31P spectrum of the DMPG-doped sample at 25 °C may be an indication that part of the sample is oriented with respect to the magnetic field.

Van Dam et al.

3284 Langmuir, Vol. 22, No. 7, 2006

Structure in Magnetically Aligning Samples. The 31P NMR spectra for the DMPG- and CTAB-doped samples between 30 and 45 °C (Figure 1) display two distinct resonances (apart from the phosphate buffer resonance at 0 ppm). This spectral appearance is indicative of magnetic alignment with the bilayer normal oriented toward being perpendicular to the magnetic field.4,9,20 The relative intensities of the two resonances, Iupfield/Idownfield, is roughly equal to the qP-value of the system (see eq 4), suggesting that the upfield (most negative chemical shift value, rightmost in the spectra) peak arises from DMPC (and DMPG) and the downfield peak from DHPC. Cryo-TEM (Figure 2c) reveals that the magnetically aligning aggregates have a structure that may be described as intermediate between a two-dimensional network built from highly branched, flattened cylindrical micelles and an extended lamellar sheet perforated by large holes. The latter description is not far from how one would describe a slice of Swiss cheese. Considerations of curvature (see, for example, ref 21) suggest that DHPC (and CTAB) should preferably cover the edges of the holes of the perforated structure and that the holes must be rather large in order to overcome the negative curvature associated with this geometry. DMPC (and DMPG) should, by curvature considerations, preferably adapt a bilayer structure and make up the bulk of the “lamellar” sheet. This morphology is consistent with the 31P NMR spectra observed (Figure 1) and with the spectral assignment of DHPC representing the smaller downfield peak and DMPC (and DMPG) the larger upfield peak. The absence of 1H NOESY cross-peaks between DMPC and DHPC observed by others under similar conditions supports a largely domainseparated aggregate morphology.22 Furthermore, it is easily seen how the flattened and branched cylindrical micelles transform smoothly first into the lacelike structures that orient in the magnetic field and eventually into a regular lamellar phase (see also Figure 7 in ref 13). The gradual upfield shift of the DMPC resonance with temperature, -0.08 ppm/°C, could be explained by an increased ordering of the perforated sheets with respect to the magnetic field of the bilayer. Increased ordering with increasing temperature has been observed in the pure DMPC/DHPC system through 2H NMR measurements of the solvent water.4 The similarly gradual but larger upfield shift of the DHPC resonance with temperature, 0.16-0.20 ppm/°C, could be explained by variations in the population of different DHPC sites in combination with the increased orientation of the aggregates. In the present system, there are three possible DHPC sites: (i) monomeric DHPC in solution, (ii) DHPC covering the edges of the holes in the perforated bilayer, and (iii) DHPC in the (DMPC-rich) bilayer. If the DHPC molecules that cover the holes of the perforated bilayer are in fast exchange with one another (most probably via aqueous monomeric DHPC), so that an average chemical shift is picked up by each molecule and the holes are isotropically distributed in the bilayer, sites i and ii should have roughly the same chemical shift. This chemical shift reads between -1.2 and -1.4 ppm, the values for monomeric and micellar DHPC, respectively (see discussion above). DHPC molecules in site iii, however, should have a chemical shift similar to that of the DMPC molecules in the bilayer, which in the absence of chemical exchange of DMPC is given by the upfield 31P resonance. (It is reasonable to believe that DMPC on the time scale of the experiment does not exchange via the solvent due to its low solubility. However, we must also assume that DMPC (20) Sanders, C. R., II.; Schwonek, J. P. Biochemistry 1992, 31, 8898-8905. (21) Israelachvili, J. N. Intermolecular & Surface Forces, 2nd ed.; Academic Press: San Diego, CA, 1991. (22) Sternin, E.; Nizza, D.; Gawrisch, K. Langmuir 2001, 17, 2610-2616.

does not partially occupy the DHPC-covered edges of the holes to any significant extent.) Under these assumptions, and if fast exchange exists between all three DHPC sites, the relative population (molar) of DHPC residing in the bilayer, pb, may be calculated from the chemical shift values in the 31P spectra in Figure 1 through

pb )

[DHPC]bilayer [DHPC]total

)

δdownfield - δiso δupfield - δiso

(5)

with [DHPC]bilayer being the concentration of DHPC in the bilayer and [DHPC]total the total DHPC concentration. δiso is the isotropic DHPC chemical shift, reading -1.2 and -1.4 ppm for DHPC in monomeric and in micellar form, respectively. It is difficult to access the precise value of δiso to use, since it depends on the relative amounts of monomeric and edge-forming DHPC, which is not accurately known. Previous investigations have shown the concentration of monomeric DHPC in these systems to be approximately 5 mM,4,13 which constitutes around 25% of the total DHPC concentration in the present samples (cL ) 5%. qX ) 3.2). This information could be used to construct a three-site model including all three sites i, ii, and iii listed above explicitly, since the total DHPC concentration is known and the chemical shift values for monomeric and for rim-forming DHPC may be estimated from the experiments on pure DHPC reported above. However, the 0.2 ppm difference between the chemical shifts for these two forms of DHPC gives only a small (e6%) difference for the outcome when calculating pb by use of eq 5, and since we do not know if micellar and rim-forming DHPC give rise to the same chemical shift value, we have chosen to use δiso ) -1.4 ppm in our calculations below, corresponding to a (admittedly unrealistic) situation where the concentration of monomeric DHPC is zero. The chemical shifts at 30 °C for the DMPG-containing sample are δdownfield ) -3.7 ppm and δupfield ) -12.0 ppm, leading, via eq 5, to pb ) 0.22, i.e., 22% of the available DHPC being situated in the bilayer of the aggregates at this temperature. Using the chemical shifts at 45 °C for the same sample, δdownfield ) -6.5 ppm and δupfield ) -13.3 ppm, respectively, results in 43% of the total amount of DHPC residing in the bilayer. For the CTABcontaining sample, the corresponding numbers are pb ) 0.20 at 30 °C and p ) 0.37 at 45 °C, respectively. The fraction of DHPC situated in the bilayer, pb, may be recast in terms of the fraction of the bilayer that is made up of DHPC, ,

)

[DHPC]bilayer [DHPC]bilayer + [DMPC] + [DMPG]

)

pb (6) p b + qP

assuming still that all of the available DMPC is in the bilayer of the aggregate. This gives  ) 0.06 and 0.12 for the DMPGcontaining sample at 30 and 45 °C, respectively. Calculations on the CTAB-containing sample give  ) 0.06 and 0.11 at the same temperatures. This means that 6-12% of the DMPC-rich bilayer is made up of DHPC, the amount increasing with increasing temperature. The approximately linear dependence of the chemical shift changes with temperature yields that the ratio  of DHPC increases approximately linearly at ∆/∆T ) 0.003-0.004/°C. The fairly small relative amount of DHPC in the DMPC-rich bilayer could escape detection by 1H-NOESY, explaining the absence of cross-peaks in the measurements carried out by Sternin et al.22 In the very recent study by Triba et al., similar numbers, as well as a linear dependence, were found at a higher total

Magnetically Aligning DMPC/DHPC Aggregates

concentration, cL ) 25%, using a more elaborate approach.19 It should be noted, however, that these authors used a mixed disk model to interpret their data. However, the intermixing interpretation of the NMR data, where DHPC is incorporated into the DMPC-rich bilayer, is not affected in principle by the choice between a discoidal and a perforated lamellar aggregate model. Yet, it is important to emphasize that there exists no direct evidence for a discoidal, magnetically aligning phase. On the contrary, experiments able to discriminate between different aggregate structures, including the cryo-TEM investigations in the present study (Figure 2), all point toward more extended aggregates, such as perforated lamellar sheets, for the magnetically aligning phase of DMPC/DHPC mixtures.9-15 Furthermore, it is hard to understand the dramatic increase in viscosity just before the onset of the aligning phase without invoking entangled cylindrical micelles, and a transformation from disks to cylindrical micelles and then back to (larger) disks appears rather unlikely. A progression from small discoidal micelles, via cylindrical micelles, on to (aligning) perforated lamellar sheets appears more realistic. It is also difficult to see how rather small disks can gain enough aggregate magnetic susceptibility anisotropy to align in NMR magnetic field strengths. The recent suggestion that it is the cylindrical micellar phase that aligns23 does not seem plausible either, since this aggregate shape makes it hard to explain the two well-separated resonances appearing in the 31P NMR spectrum (Figure 1). Furthermore, it clearly seems to be the case that an increase in the order parameter of the aligning phase correlates with a decrease in sample viscosity (Figure 1 and ref 13), rendering individual cylindrical micelles, which constitute the viscous phase,13 unlikely to be the magnetically aligning aggregates. Taken together, the above arguments very strongly suggest that the magnetically aligning phase of DMPC/DHPC consists of perforated lamellar aggregates and not of disks or cylindrical micelles. Effects Observed with Increasing Temperature. The effect of the gradual transfer of DHPC to the DMPC-rich bilayer is to decrease either the size of the holes in the bilayer sheet or the number of holes and thus to increase the area of the bilayer. This is likely to increase, in turn, the alignability of the aggregates, since the aggregate magnetic susceptibility anisotropy increases as the holes decrease. An increased alignability with increasing temperature is in line with the experimentally observed upfield shift of the DMPC resonance with increased temperature (Figure 1), as well as with observations made using 2H NMR.4 (It should be noted here that this conclusion does not depend on the gradual upfield shift of the DMPC resonance, since it is much smaller than the gradual upfield shift of the DHPC resonance.) An effect on the 31P chemical shifts similar to that which we observe with increasing temperature (Figure 1) was seen also with increasing DMPC/DHPC ratio q,19,20 which seems natural if increased ordering is due to the smaller amount, or size, of holes in the bilayer. At 50 °C, both the DMPG- and CTAB-doped systems display the appearance of a 31P spectral line at -1.4 ppm, as well as a diminishing and broadening of the two resonances arising from the oriented phase (see top row of Figure 1). Similar spectra have (23) Harroun, T. A.; Koslowsky, M.; Nieh, M.-P.; de Lannoy, C.-F.; Raghunathan, V. A.; Katsaras, J. Langmuir 2005, 21, 5356-5361.

Langmuir, Vol. 22, No. 7, 2006 3285

been observed by others at high temperatures.4,19 The chemical shift of the new resonance, -1.4 ppm, is indicative of isotropically distributed phosphatidylcholines in some sort of aggregate, possibly (probably) in exchange with a population of monomeric phosphatidylcholine. These aggregates could be small globular or discoidal micelles of the same kind as those observed below the DMPC transition temperature (see the bottom row of Figure 1 and also Figure 2a). Alternatively, the resonance at -1.4 ppm could result from DHPC covering the edges of holes in isotropically distributed DMPC-based aggregates. Our previous study of the pure DMPC/DHPC system showed that large multilamellar vesicles (LMV’s) were formed at high temperature at q ) 3.13 LMV’s cannot align in magnetic fields for symmetry reasons (i.e., because they are virtually spherical) and the holes in such aggregates would thus be isotropically distributed. The presence of large LMV’s is expected to give rise to a DMPC spectrum in the shape of a powder pattern, since the aggregates are too large for motional averaging to occur on the NMR time scale. A pure DMPC dispersion at 30 °C displayed a 31P spectrum in line with the expected powder pattern,20 where we only observe signal close to, and peaking at, the 90° component at roughly -17 ppm (data not shown). No spectral intensity appears, however, at -17 ppm in the DHPC/DMPC-based mixtures at 50 °C, which could simply be due to the low intensity of this resonance (since it is distributed over a large part of the spectrum). The simultaneous presence of an oriented phase at 50 °C, manifested through the two high-field resonances at roughly -8 and -15 ppm in the spectra (top row of Figure 1), indicates that two phases are present in the sample at this temperature, i.e., that phase separation has occurred. The very high chemical shift values of the peaks indicate a highly oriented phase. Further, both high-field resonances become considerably broader when going from 45 to 50 °C. This could reflect a decrease in the dynamics of the aligned phase, or it could reflect a decrease in NMR magnetic field homogeneity due to a macroscopic phase separation in the NMR tube.

Conclusions The results of the present study confirm that the magnetically aligning phase of DMPC/DHPC mixtures consists of extended aggregates that are best described as intermediates between twodimensional networks of branched flattened cylindrical micelles and highly perforated lamellar sheets. The presence of two distinct 31P NMR resonances, roughly corresponding to the DMPC/DHPC ratio, suggests that the bulk of the perforated bilayer is composed of DMPC, while DHPC preferentially covers the edges of the holes. However, 20-43% of the DHPC is situated in the flat bilayer part of the aggregate, the number increasing approximately linearly with increasing temperature. This explains the increasing degree of alignment of the aggregates with increasing temperature, since the magnetic susceptibility anisotropy of the aggregate builds up as the bilayer grows. Acknowledgment. We are most grateful to Dr. Adolf Gogoll for generously providing us access to the NMR spectrometer. This research was financially supported by the Swedish Research Council (Vetenskapsrådet). LA052988M