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Temperature-Composition Diagram of Dimyristoylphosphatidylcholine-Dicaproylphosphatidylcholine “Bicelles” Self-Orienting in the Magnetic Field. A Solid State 2H and 31P NMR Study Ge´rard Raffard,† Siegfried Steinbruckner,† Alexandre Arnold,‡ James H. Davis,§ and Erick J. Dufourc*,†,‡ Centre de Recherche Paul Pascal, CNRS, Bordeaux-Pessac, France, Department of Physics, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and Institut Europe´ en de Chimie et Biologie, Ecole Polytechnique, Bordeaux-Pessac, France Received April 14, 2000. In Final Form: June 26, 2000 Mixtures of dimyristoylphosphatidylcholine (DMPC) and dicaproylphosphatidylcholine (DCPC) were investigated by solid-state 31P and 2H NMR. By variation of the mole fraction, X, of DMPC in DCPC, the temperature, and the water content, the conditions at which small discoidal particles, the so-called “bicelles”, self-orient in the magnetic field were determined. The bicellar region has an ellipsoidal shape and is delineated by compositions and temperatures ranging from X ) 65-87% and T ) 25-45 °C, at 80% hydration in the presence of 100 mM KCl. In the absence of salt, the bicelles still orient in the field but for a narrower range of composition (X ≈ 72-87%). The degree of macroscopic orientation increases in the presence of salt. Reducing hydration to 40% increases the breadth of the bicelle domain. On the contrary, above 95% water content, bicelles are no longer detected by NMR. In the bicelle domain, water shows residual ordering which linearly increases with the decrease in water content. Analysis of the water behavior in terms of bound and free water suggests that water bound to bicelles has the same surface properties as in pure lipid membranes and that it promotes swelling. Perdeuteration of the DMPC lipid chains indicates a reduction in local ordering in the bicelle core, relative to pure lipid dispersions, for corresponding temperatures. As a consequence, the bilayer would be slightly thinner in the bicelle.
Introduction It has been reported that some biological membranes such as bacteriorhodopsin thylacoid or E. coli membranes1 may show some degree of macroscopic orientation in magnetic or electric fields. The general observation is, however, the converse, biological membranes or lipid extracts do not usually orient appreciably in a magnetic field. This may represent a drawback when one wishes to perform NMR experiments on hydrated lipid-protein systems. The only alternative is to perform broad line experiments on powder-like systems or use magic angle spinning (MAS) techniques to measure the isotropic information on chemical shifts or coupling constants. The orientational information, such as rigid body orientation of peptidic helices or order parameters of flexible chains, may still be obtained from samples macroscopically oriented between glass plates. An alternative to these experiments is to use self-orienting synthetic membranes. Such systems have been discovered after the pioneering works of Roberts2-6 and Sanders.7-10 They are made of binary mixtures of long-chain (C14-C18) and short-chain * To whom correspondence may be addressed. IECB-Polytechnique, Av. Pey Berland, BP 108, 33402 Talence cedex, France. Tel. and Fax: (33) +5 57 96 22 18. E-mail: erick.dufourc@ iecb-polytechnique.u-bordeaux.fr. † Centre de Recherche Paul Pascal. ‡ Institut Europe ´ en de Chimie et Biologie. § University of Guelph. (1) Seelig, J.; Borle, F.; Cross, T. A. Magnetic ordering of phospholipid membranes. Biochim. Biophys. Acta 1985, 814, 195-198. (2) Gabriel, E. N.; Roberts, M. F. Spontaneous formation of stable Unilamellar Vesicles. Biochemistry 1984, 23, 4011-4015. (3) Gabriel, E. N.; Roberts, M. F. Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesiscles that form spontaneously. Biochemistry 1986, 25, 2812-2821.
(C6-C8) phospholipids or bile salts analogues and are supposed to take the form of disk-shaped bilayer micelles (with a diameter of 100-1000 Å and the thickness of a lipid bilayer, 40-60 Å).3,5 These so-called “bicelles” are able to orient themselves with the normal to the disk perpendicular to the magnetic field. Change in their orientation, i.e., normal to the disk parallel to the magnetic field, can be induced by adding aromatic amphiphiles such as naphthol10 or paramagnetic ions such as Eu3+, Er3+, Tm3+, and Yb3+,11-13 making the bicelle a very attractive “biological goniometer”. Successful insertion of some membrane proteins into bicelles has been accomplished.14 (4) Gabriel, E. N.; Roberts, M. F. Short-chain lecithin/longchain phospholipid unilamellar vesicles: asymmetry, dynamics, and enzymatic hydrolysis of the short-chain component. Biochemistry 1987, 26, 2432-2440. (5) Eum, K. M.; Riedy, G.; Langley, K. H.; Roberts, M. F. Temperatureinduced fusion of small unilamellar vesicles formed from saturated long-chain lecithins and diheptanoylphosphatidylcholine. Biochemistry 1989, 28, 8206-8213. (6) Bian, J.; Roberts, M. F. Phase separation in short-chain lecithin/ gel-state long-chain lecithin aggregates. Biochemistry 1990, 29, 79287935. (7) Sanders, C. R.; Prestegard. J. H. Magnetically oreientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO. Biophys. J. 1990, 58, 447-460. (8) Sanders, C. R. I.; Prestegard, J. H. Headgroup orientations of alkyl glycosides at a lipid bilayer interface. J. Am. Chem. Soc. 1992, 114, 7096-7107. (9) Sanders, C. R.; Schwonek, J. P. Characterization of magnetically orientable bilayers in mixtures of dihexanoylphophatidylcholine and dimytistoylphosphatidylcholine by solid-state NMR. Biochemistry 1992, 31, 8898-8905. (10) Sanders, C. R.; Schaff, J. E.; Prestegard, J. H. Orientational behavior of phosphatidylcholine bilayers in the presence of aromatic amphiphiles and a magnetic field. Biophys. J. 1993, 64, 1069-1080. (11) Prosser, S. R.; Hwang, J. S.; Vold, R. R. Magnetically aligned phospholipids bilayers with positive ordering: a new model membrane system. Biophys. J. 1998, 74, 2405-2418.
10.1021/la000564g CCC: $19.00 © 2000 American Chemical Society Published on Web 09/06/2000
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For a review see Sanders and Prosser.15 Unfortunately, not all membrane proteins can be reconstituted in such a way. In some cases the bicelle is no longer stable, making it impossible to magnetically orient the protein. To better understand the stability and orientational properties of the bicelles, we have investigated the binary phase diagram (as a function of composition and temperature) of dimyristoylphosphatidylcholine (DMPC, C14PC) mixed with dicaproylphosphatidylcholine (DCPC, C6PC). The diagram has been determined by static solid state 31P and 2H NMR, which makes it possible to identify the lipid phases16,17 and to distinguish micelles from oriented bicelles and from nonoriented lipid dispersions. Phase boundaries between which the self-orienting bicelles exist can thus be delineated. It must be mentioned that a similar diagram on C16/C7 phospholipid mixtures has been determined mainly by differential scanning calorimetry (DSC) and fluorescence.6 The bicelles appear to exist between 0.9 and 0.65 mol fraction of C16PC in C7PC. Unfortunately, the concentration and temperature ranges for self-orientation in the magnetic field had not been determined. Interestingly, the bicelles have also been used to partially orient water-soluble proteins. The latter that have been dissolved in such aqueous liquid crystalline medium exhibit partial ordering properties that can be used to gain extra structural information for solving the protein 3D structure.18,19 We have, therefore, investigated the influence of water content on the C14/C6 phase diagram as well as the presence of KCl. The present study reports for the first time formal phase diagrams (temperature-composition and hydration) of lipids mixtures that form so-called bicelles self-orienting in magnetic fields. It demonstrates also that 100 mM KCl greatly improves both the formation and the orientation of bicelles in the magnetic field. Materials and Methods Chemicals. Synthetic DMPC, DCPC, and {sn-2-2H27}-DMPC were purchased from Avanti Polar Lipids (USA) and used without further purification. D2O and deuterium-depleted water were obtained from Eurisotop (France). Possible hydrolysis of lipids was checked after completion of NMR experiments by thin-layer chromatography. Less than 5% lysolipid was detected. Sample Preparation. DMPC and DCPC powders were weighed in appropriate quantities, such that the molar content of DMPC vs the total lipid ranged from 100% (pure C14PC) to 0% (pure C6PC), and were mixed together in a centrifuge vial. To 25 (12) Prosser, S. R.; Hunt, S. A.; DiNatale, J. A.; Vold, R. R. Magnetically aligned membrane model systems with positive order parameters: switching the sign of Szz with paramagnetic ions. J. Am. Chem. Soc. 1996, 118, 269-270. (13) Katsaras, J.; Donaberger, R. L.; Swainson, I. P.; Tennant, D. C.; Tun, Z.; Vold, R. R.; Prosser, R. S. Rarely observed phase transitions in a novel lyotropic liquid crystal system. Phys. Rev. Lett. 1997, 78, 899-902. (14) Sanders, C. R.; Landis, G. C. Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 1995, 34, 4030-4040. (15) Sanders, C. R.; Prosser, R. S. Bicelles: a model membrane system for all seasons? Structure 1998, 6, 1227-1234. (16) Marinov, R.; Dufourc, E. J. Cholesterol stabilizes the hexagonal type II phase of 1-palmitoyl-2-oleoyl sn glycero-3-phosphoethanolamine. A solid state 2H and 31P NMR study. J. Chim. Phys. 1995, 92, 17271731. (17) Marinov, R.; Dufourc, E. J. Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state 2H and 31P NMR. Eur. Biophys. J. 1996, 24, 423-431. (18) Bax, A.; Tjandra, N. High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid cristalline medium. J. Biomol. NMR 1997, 10, 289-292. (19) Tjandra, N.; Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 1997, 278, 1111-1114.
Raffard et al. mg of total lipids was added the amount of water necessary to obtain hydration (w/w) ranging from 95% to 40%. The vial was sealed with Teflon tape. The following cycle of sample equilibration was then performed: vigorous vortexing; freezing in liquid nitrogen for 2 min; warming up in a 50 °C water bath for 10 min; 10 min of centrifugation at 3000 rpm (radius from spindle to tube of 150 mm, 1500g). This cycle was repeated five times. Evidence for bicelle formation was obtained by inspection of sample by eye. Samples in the bicellar region, as detected by NMR, formed a highly viscous, transparent gel. NMR Spectroscopy. NMR was carried out on Bruker ARX 300, MSL 200, and DRX 400 machines. 31P NMR spectra were acquired using a phase-cycled Hahn-echo pulse sequence with gated broad band proton decoupling.20 A deuterium (D2O) lock was used, and 1k-2k acquisitions were recorded. 2H NMR experiments on labeled lipids were performed by means of a quadrupolar echo composite pulse sequence21,22 and typically 10 000 scans. 2H NMR spectra on D2O were achieved using a phase-cycled quadrupolar echo pulse sequence23 with proton spin lock decoupling, typically recording a few hundred scans. Typical acquisition parameters were as follows: spectral window of 50 kHz for 31P NMR and 250 kHz and 10 kHz for 2H NMR on labeled phospholipid and D2O, respectively; π/2 pulse widths ranged from 4 to 8 µs depending on spectrometers, interpulse delays were 40-50 µs. A recycle delay of 6-8 s was used for 31P NMR; for 2H NMR experiments it was set to 2 s. 31P chemical shifts were expressed relative to 85% H3PO4 (0 ppm). Quadrature detection was used in all cases. Samples were allowed to equilibrate at least 30 min at a given temperature before the NMR signal was acquired; the temperature was regulated to (1 °C. All thermal variations were performed first by decreasing the temperature. It was, however, checked that increasing the temperature gave the same results, provided a 30-min equilibrium time was used.
Results 31P NMR Spectroscopy of DMPC-DCPC Systems.
Samples with mole fractions of DMPC ranging from X ) 100% to 0% were made with 80% D2O content in the presence of KCl (0.1 M). The temperature was varied from 60 to 5 °C. Spectra for X lower than 66% all exhibit a single sharp line centered at -0.4 ppm, the isotropic chemical shift of phosphatidylcholines, reflecting the presence of micelles whatever the temperature. Above X ) 90% the spectrum is composed of a single axially symmetric powder pattern of ∆σ ≈ 45 ppm characterizing an unoriented lamellar phase at all temperatures. Selected spectra for molar fractions between these two boundaries (X ) 66%, 75%, and 90%) are shown in Figure 1 and display a wide variety of shapes: (i) axially symmetric powder patterns: (ii) two sharp lines with chemical shifts of about -4 ppm and -10 ppm; (iii) powder patterns with superimposed sharp lines of isotropic chemical shift; (iv) single isotropic sharp lines. Cases i and iv can be attributed to a lamellar phase and micelles, respectively, whereas (iii) can be attributed to a mixture of micelles and lamellar phases and the two single lines, situation (ii), reflect the macroscopic orientation of bicelles with the normal to the bilayer perpendicular to the magnetic field.9 31P Chemical Shift Temperature Dependence. The above characteristic spectra can be used to delineate the phase boundaries between which bicelles self-orient in the magnetic field. In this respect, the chemical shifts of (20) Rance, M.; Byrd, R. A. Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media: Phase-Cycled Hahn echo spectroscopy. J. Magn. Reson. 1983, 52, 221-240. (21) Levitt, M. H.; Freeman, R. Compensation for pulse imperfections in NMR spin-echo experiments. J. Magn. Reson. 1981, 43, 65-80. (22) Levitt, M. H. Symmetrical composite pulse sequences for NMR population inversion. I. Compensation of Radio frequency field inhomogeneity. J. Magn. Reson. 1982, 48, 234-264. (23) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. I.; Higgs, T. P. Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem. Phys. Lett. 1976, 42, 390-394.
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Figure 1. Proton-decoupled 31P NMR spectra of DMPC and DCPC mixtures in 80% (w/w) D2O. Mole fractions X ) [DMPC]/ ([DMPC] + [DCPC]) are 90% (left), 75% (middle), and 66% (right). Temperature is indicated on spectra. NMR signals were acquired with the Hahn echo sequence, and deuterium lock was used during acquisitions. Number of acquisitions ranged from 1k to 2k, and a line broadening of 10 Hz was applied prior to Fourier transformation. Chemical shifts are expressed relative to 85% H3PO4 (0 ppm).
the different spectral features can be used. Figure 2 displays the thermal variation of 31P chemical shifts for all the lines that can be detected for a sample with an 81% DMPC content. Depending on the temperature, up to three chemical shifts can be observed at once. At low temperature only the isotropic chemical shift can be detected. Upon a temperature increase, its value slightly increases,
then the line disappears and reappears at high temperatures where the chemical shift value still increases. Its temperature dependence is linear. The two chemical shifts characterizing DCPC and DMPC in bicelles decrease from -2 to -5 ppm and from -8 to -12 ppm, respectively, as the temperature increases. The peak standing for the 90° orientation of an axially symmetric powder pattern is
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Figure 2. Temperature dependence of 31P chemical shift for all observed spectral features in the system DMPC/DCPC, X ) 81%: (9) isotropic line; (2) DMPC in bicelle; (b) DCPC in bicelle; (1) peak for the 90° orientation in axially symmetric powder pattern. Solid lines represent a linear fit, whereas vertical dashed lines are drawn to help in delineating phase boundaries. One-phase regions, as detected by NMR, are denoted I, B, and L for isotropic, bicellar, and lamellar. Figure 4. Proton-decoupled 31P NMR spectra of DMPC and DCPC mixtures in 80% (w/w) D2O, in the presence and absence of 100 mM KCl: (A and B), spectra at 33 and 36 °C without KCl; (C and D), spectra at 33 and 36 °C with KCl. NMR acquisition parameters are as in Figure 1. Mole fraction is X ) 78%. Spectra are plotted in absolute vertical scale.
Figure 3. Temperature-composition diagram of DMPC/DCPC in 80% (w/w) D2O, 100 mM KCl, as determined by 31P NMR: (2) single phase of bicelles self-orienting into the magnetic field; (1) self-orienting bicelles coexisting with isotropic or unoriented phases; (O) unoriented plus isotropic phases; (0) isotropic phase (micelles); (]) lamellar phase. Mole fractions X ) [DMPC]/ ([DMPC] + [DCPC]) are expressed in percent. One-phase regions are denoted I, B, and L for isotropic, bicellar, and lamellar phases. Solid and dashed lines are tentatively drawn to help viewing phase boundaries.
observed for temperatures below and above those for which bicelles appear. For elevated temperatures, its temperature dependence is linear. Figure 2 shows how the chemical shift can be used to identify the three singlephase regions (denoted I, B, and L for isotropic, bicellar, and lamellar) and the boundaries between them as the temperature is varied. Two-phase regions are observed between isotropic and bicellar phases and bicellar and lamellar domains. It must be mentioned that the above chemical shift variations are quite general for all compositions. Temperature-CompositionDiagramoftheDMPCDCPC System, with 100 mM KCl. By use of the 31P NMR chemical shift and characteristic spectral line shapes, the temperature-composition diagram of DMPC/ DCPC can be constructed (Figure 3). Again, single-phase regions are denoted as I, B, and L. For 62% DMPC content and below, a single isotropic phase that can be assigned
to mixed micelles is detected. This phase appears to extend to higher percentages of DMPC for temperatures lower than 20 °C. The region in which bicelles self-orient in the magnetic field is roughly delineated by compositions and temperatures ranging from X ) 65 to 87% and T ) 25 to 45 °C. The extended lamellar phase is observed for compositions above X ) 90% and/or high temperatures. Two-phase regions are observed between single-phase regions. They are composed of either bicelles coexisting with lamellar or isotropic phases or a lamellar phase with an isotropic phase. 31 P NMR Spectroscopy in the Absence of Salt. All the series of experiments have been performed again in the absence of 100 mM KCl. Basically the same spectral features, i.e., the same phases, are observed. There are, however, marked differences both in concentration and temperature ranges over which phases occur and in the shape of lines in the bicelles domain. Figure 4 shows spectra in the region where pure bicelles are observed, for X ) 78%. Lines are clearly much broader in the absence of salt. They also depart from isotropic line shape and show residual powder orientational distribution. Chemical shifts measured at sharp peaks in the absence of salt are essentially the same, within experimental error, as those measured when the sample contains KCl. The same procedure for phase boundary determination, as described above, was followed to build the temperature-composition diagram in the absence of salt, Figure 5. This diagram is very similar to what is observed with KCl. One detects lamellar, bicellar, and micellar one-phase regions and also two-phase regions. The composition range for which bicelles self-orient in the magnetic field is smaller, however, since it only spans from X ≈ 72 to 87%. The temperature range is basically unchanged. 2H NMR Spectroscopy of Lipid Chains. Solid-state deuterium NMR was performed on a system containing 75% DMPC with the sn-2 chain perdeuterated, in the presence of 100 mM KCl. The temperature variation of
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Langmuir, Vol. 16, No. 20, 2000 7659 Table 1. Carbon-Deuterium Order Parameters of Bicelles and Lipid Dispersions
Figure 5. Temperature-composition diagram of DMPC/DCPC in 80% (w/w) D2O as determined by 31P NMR: (2) single phase of bicelles self-orienting into the magnetic field; (1) self-orienting bicelles coexisting with isotropic or unoriented phases; (O) unoriented plus isotropic phases; (0) isotropic phase (micelles); (]) lamellar phase. Mole fractions X ) [DMPC]/([DMPC] + [DHPC]) are expressed in peercent. One-phase regions, are denoted I, B, and L for isotropic, bicellar, and lamellar phases. Solid and dashed lines are tentatively drawn to help viewing phase boundaries.
Figure 6. Temperature dependence of 2H NMR spectra of sn-2 perdeuterated DMPC embedded in a DMPC/DCPC system (X ) 78%), 100 mM KCl. Temperature is indicated on spectra. NMR signals were acquired with the quadrupolar echo composite pulse sequence. Numbers of acquisitions were of the order of 10k, and a line broadening of 100 Hz was applied prior to Fourier transformation. The isotropic line was arbitrarily assigned to 0 Hz.
some spectra is shown in Figure 6. At 25 °C one observes a powder pattern, where all methyl and methylene Pake patterns are superimposed, which is characteristic of a fluid phase, although with broader lines. A small sharp line also appears at the isotropic frequency. These two features indicate the presence of unoriented bilayers and a small proportion of isotropic phase (micelles). On increase of the temperature, spectra become more resolved and at
ka
DMPCb
DMPC in bicellesc
∆S/S (%)d
DMPC in bicelles/ Sbilayer ) 0.737e
2S 2R 3 4 5 6 7 8 9 10 11 12 13 14
0.094 -0.141 -0.212 -0.220 -0.220 -0.222 -0.216 -0.210 -0.198 -0.176 -0.161 -0.139 -0.111 -0.027
0.070 -0.103 -0.157 -0.167 -0.167 -0.167 -0.167 -0.157 -0.157 -0.143 -0.131 -0.110 -0.088 -0.021
25.5 26.9 25.9 24.1 24.1 24.8 22.7 25.2 20.7 18.7 18.6 20.9 20.7 22.2
0.095 -0.140 -0.213 -0.227 -0.227 -0.227 -0.227 -0.213 -0.213 -0.194 -0.178 -0.149 -0.119 -0.028
a Labeled carbon position. b DMPC as water dispersion (large multilamellar vesicles). Data from DMPC labeled on the sn-2 chain, from Douliez et al.,24 35 °C. c DMPC in DMPC-DCPC bicelles (75% DMPC, 80% hydration, 100 mM KCl, 35 °C). d (SDMPC - SDMPCbicelles)/ SDMPC. e Same as in footnote c but divided by Sbilayer (order parameter associated to the bilayer overall motion (see text), 2S 2S 2R 2R Sbilayer ) {(SDMPCbicelles /SDMPC ) + (SDMPCbicelles /SDMPC )}/2).
35 °C a well-defined pattern characteristic of an oriented sample with its bilayer normal oriented at 90° with respect to the magnetic field is observed. Nine quadrupolar splittings can easily be measured. On increase of the temperature further, one obtains at 50 °C a superimposition of powder patterns and an isotropic line. All the observed spectral features are in complete agreement with the 31P NMR data. The quadrupolar splittings, ∆νQ, were measured at 35 °C, and the corresponding SCD order parameters were calculated and are reported in Table 1, together with corresponding data on extended bilayers (pure DMPC in lamellar LR phase) from Douliez et al.24 The following equation was used: |SCD| ) 4∆νQ/3AQ, where AQ ) 167 kHz is the quadrupolar coupling constant for methylene bonds.25 Attribution and sign determination were made on the basis of extended bilayers. Table 1 also reports the relative variation of order parameters with respect to DMPC. Figures indicate a SCD reduction from 18.6% to 26.7% depending upon labeled carbon position. No straightforward relation can be deducted on going from the interface down to the bilayer center. 31 P NMR and 2H NMR as Function of Hydration. Experiments were performed on the sample containing 78% DMPC and 100 mM KCl, as a function of hydration and temperature. D2O content varied from 40 to 95%, and temperatures ranged from 20 to 60 °C. Hydration is defined as the mass of water over the total mass (lipid + water). General spectral features as already reported were detected, in particular the presence of two sharp lines in the 31P spectra, at about -4 and -10 ppm, indicative of bicelles self-orienting in the magnetic field. From these oriented spectra, it was possible to delineate the boundaries of the domain in which bicelles appear to self-orient in the magnetic field. Outside of these phase boundaries, two-phase regions are observed. The data afforded the construction of a temperature-hydration diagram, Figure 7. It clearly appears that reducing hydration increases the breadth of the bicelle domain. On the other hand, (24) Douliez, J. P.; Le´onard, A.; Dufourc, E. J. A restatement of order parameters in biomembranes. Calculation of C-C bond order parameters from C-D quadrupolar splittings. Biophys. J. 1995, 68, 1727-1739. (25) Burnett, L. J.; Mu¨ller, B. H. Deuteron quadrupolar coupling constants in three solid deuterated paraffin hydrocarbons: C2D6, C4D10, C6D14. J. Chem. Phys. 1971, 55, 5829-5831.
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Figure 7. Temperature-hydration domain of existence for self-orienting bicelles (X ) 78%, 100 mM KCl). B denotes the region for which pure self-orienting bicelles are detected by 31P NMR as two sharp lines. Upper and lower phase boundaries are reported with an accuracy of ca. 2 °C in temperature and 2% in hydration. Solid lines are drawn to help in viewing phase boundaries.
Figure 8. D2O quadrupolar splitting as a function of water content (w/w) in DMPC/DCPC mixtures (X ) 78%, 100 mM KCl, T ) 38 °C). The solid line is a least-squares fit of a linear function.
above 95% water, bicelles are no longer detected by NMR. Because samples were prepared in D2O, it was easy to observe the structure and dynamics of water using 2H NMR. Spectra consist of a quadrupolar doublet except for 95% water content for which the doublet collapsed into an isotropic line. Quadrupolar splittings are plotted in Figure 8 versus hydration. They all fall on a straight line, within the experimental error. Discussion The results section clearly brings information on both macroscopic and microscopic properties on short-chain to long-chain lipid mixtures. Phase diagram, effect of salt, hydration, and order in the bilayer and in the water layer are obtained. These many aspects will be discussed in the following. Macroscopic Properties. Phosphorus NMR was proven to be a nice tool in leading to the construction of temperature-composition diagrams. It clearly appears that the disklike membranous objects, so-called bicelles, self-orient between 65 and 87% DMPC in the system, for temperatures ranging from 25 to 45 °C and for 80% water. The pure bicelle domain shows an ellipsis shape with a
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widest span in temperature for X ≈ 75-80%. Surrounding this one-phase domain, one finds bicelles coexisting with either a lamellae or isotropic (from the NMR point of view) phase. The two-phase regions deserve a comment. It is clear that these regions are complex and contain phase boundaries and triple points. DSC experiments could be performed in these two-phase regions to understand better the phase diagram of DMPC/DCPC. Further away from the domain are the lamellar phospholipid phase (high DMPC concentrations and/or high temperatures) and the isotropic phase (low DMPC content and/or low temperatures). It is interesting to compare our diagram with that reported by Bian and Roberts6 on a similar system: dipalmitoyl-PC/diheptanoyl-PC. The authors report a domain where small bilayer particles appear for X ranging from 90% to 55-70%. This domain gets narrower in composition when the temperature increases above 40 °C. This agrees with our findings as far as lipid composition is concerned. They also report “classical” mixed micelles for X < 60% and multibilayers for X > 90%, which also agrees well with our isotropic and lamellar phases. It is interesting to detail the domain where bilayer particles are encountered. These latter are reported to have approximately a 90 Å hydrodynamic radius at low temperatures and to fuse to larger structures at 38-40 °C.4 This finding is fully compatible with the appearance of the transition from an isotropic phase at low temperatures to our bicellar domain at about 25-30 °C (with DMPC instead of DPPC). At this level of discussion it is important to note that the viscosity of the sample greatly changes as a function of temperature. As can be readily seen by eye, samples for X ≈ 65-87% flow in the NMR tube at low (0-20 °C) temperatures whereas a highly viscous gel is formed around 25 °C and above in full agreement with viscosity measurements.26 From the NMR point of view, small objects undergoing fast isotropic tumbling due to the low viscosity of the medium will give rise to an isotropic line, even if they have a discoidal shape. In other words, it is not possible to tell from NMR spectra whether the isotropic lines seen for X ≈ 65-87% and T < 20 °C reflect the presence of mixed micelles or small discoidal objects. However the region where objects selforient in the field is clearly evident from our experiments. Above 50 °C spectra reflect the presence of isotropic plus powder pattern line shapes. The powder pattern can be attributed to nonoriented material, whereas it is not possible to tell whether the isotropic line is from bicelles, which can no longer orient in the field and tumble, or to micelles. It is interesting to note that, in the absence of KCl, the center of the elliptical domain is also found for X ≈ 80%, T ) 35 °C. So the salt is not necessary for bicelles formation, but it appears to extend their domain of existence toward the short-chain lipid side. The temperature range is unchanged in the absence of salt. Also of importance is the effect of hydration on the breadth of the temperature range of the bicellar phase region. Figure 7 indicates that dehydration stabilizes the bicelles. This is clearly linked to a tighter packing of the bicelles (lesser swelling) concomitant with a greater cooperativity of the objects for keeping their alignment with the field. Another interesting effect of KCl is the induction of a better orientation of the system with respect to the magnetic field (Figure 4). Here it must be recalled that the headgroup may change its orientation with respect to (26) Struppe, J.; Vold, R. R. Dilute bicellar solutions for structural NMR work. J. Magn. Reson. 1998, 135, 541-546.
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the bilayer normal upon addition of salts. This has been extensively studied by Seelig and others especially in the case of negatively charged lipids.27 However, the 31P chemical shifts are essentially unchanged in the presence of KCl. This strongly suggests that the line sharpening detected with KCl cannot be attributed to a change in phosphate orientation but rather to an increase in the degree of orientation in the magnetic field. Unfortunately, we have little data on the possible localization of the salt with respect to the lipid interface to ascertain this hypothesis. Microscopic Properties. In the bicellar region, X ) 75%, the quadrupolar splittings for DMPC are nicely resolved. Their position corresponds to that of a bilayer normal oriented perpendicular to the magnetic field, confirming the 31P NMR results. As a side comment, the oriented deuterium spectrum no longer requires the spectral deconvolution, so-called de-Paking, as introduced by Davis and Bloom28,29 to resolve multiple splittings in powder spectra. Quadrupolar splittings provide a measure of the orientational ordering of C-D bonds embedded in anisotropic media, SCD. Such an order parameter depends on several averaging modes provided by intramolecular (Sintra), intermolecular (Sinter), and collective motions (Scoll).24,30-33 This description has been applied to the bicelles by Sanders and Schwonek9 and the corresponding order parameters called Sloc, Smol, and Sbilayer. The latter authors further assigned the SCD decrease as observed in bicelles to the onset of overall bilayer motions. They propose a scaling factor, in fact Sbilayer, which would account for this disordering. Following Sanders and Schwonek, who state that the average conformation, dynamics, and orientation of the headgroup, glycerol backbone, and R-carbon regions of DMPC are unperturbed by the addition of DCPC, one may estimate Sbilayer from positions 2R and 2S
Sbilayer ) 2S 2R 2R {(SDMPCbicelles /S2S DMPC) + (SDMPCbicelles/SDMPC)}/2
Table 1 thus reports order parameter values for DMPC in bicelles after correction of whole bicelle wobbling by Sbilayer. It is clearly noticed that figures are markedly higher than those found for pure DMPC, independently of the labeled position. This would suggest that the short-chain lipid orders DMPC in the bicelle, which is rather difficult to account for. Alternatively, one could take the opposite hypothesis, i.e., bicelle reorientation as a whole is negligible, and then assign the marked apparent disordering to the presence of the detergent-like lipid, DCPC. In both (27) Scherer, P. G.; Seelig, J. Electric charge effect on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles. Biochemistry 1989, 28, 7720-7728. (28) Bloom, M.; Davis, J. H.; Mackay, A. L. Direct determination of the oriented sample NMR spectrum for systems with local axial symmetry. Chem. Phys. Lett. 1981, 80, 198-201. (29) Sternin, E.; Bloom, M.; MacKay, A. L. De-Pake-ing of NMR Spectra. J. Magn. Reson. 1983, 55, 274-282. (30) Petersen, N. O.; Chan, S. I. More on the motional state of lipid bilayer membranes: Interpretation of order parameters obtained from nuclear magnetic resonance experiments. Biochemistry 1977, 16, 2657-2667. (31) Meier, P.; Ohmes, E.; Kothe, G. Multipulse dynamic nuclear magnetic resonance of phospholipid membranes. J. Chem. Phys. 1986, 85, 3598-3614. (32) Dufourc, E. J.; Mayer, C.; Stohrer, J.; Althoff, G.; Kothe, G. Dynamics of phosphate head groups in biomembranes. A comprehensive analysis using phosphorus-31 nuclear magnetic resonance lineshape and relaxation time measurements. Biophys. J. 1992, 61, 42-57. (33) Sto¨hrer, J.; Gro¨bner, G.; Reimer, D.; Weisz, K.; Mayer, C.; Kothe, G. Collective lipid motions in bilayer membranes studied by transverse deuteron spin relaxation. J. Chem. Phys. 1991, 95, 672-678.
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hypotheses the ordering information can be translated in terms of average chain length as recently proposed.24,34-36 By doing so, it is found that the sn-2 chain has average lengths of 11.5, 10.9, and 11.6 Å in pure DMPC and in bicelles without and with the Sbilayer correction, respectively. Accuracy in chain length calculation depends on accuracy in SCD measurement, which is better that 1%. As a conclusion, if the whole bicelle motion accounting for all the apparent chain disordering, this would lead to a bilayer membrane thickness slightly larger or comparable to that of pure DMPC. Alternatively if this motion is absent, the bilayer hydrophobic thickness would be some 1.2 Å thinner in the bicelle structure. It is clearly hard to decide which hypothesis is correct based on the available data. It must however be mentioned that collective motions have been observed to promote a decrease in quadrupolar splittings when diluting samples,37-39 and in this respect the bicelle sample is more concentrated than the pure lipid sample. Also, bicelle tumbling would induce line broadening; this is not observed here. Hence, and without discarding completely the whole bicelle motion, we tend to favor the DCPC local disordering effect for our set of data. The short-chain lipid would thus perturb the packing of the DMPC chains and hence promote larger degrees of freedom for the entire bilayer core. It must however be kept in mind that the effect is limited and does not markedly modify the bilayer properties of the bicelle. The water behavior is of interest in the bicellar region because it is no longer isotropic. A single doublet is resolved of residual quadrupolar splitting varying linearly from 0 Hz at 95% hydration to 200 Hz at 60% hydration. Values for lower hydration were very difficult to measure and are not reported. It is interesting at this level of discussion to recall the behavior of water in a pure DMPC system as the hydration is varied.40 In the fluid phase, nD ()10) water molecules per headgroup are used to constitute the first hydration shell. Under these conditions, water is tightly bound to lipids with a quadrupolar splitting, ∆νD, of 1200 Hz. On increase of hydration above 10 water molecules per lipid, a decrease of the quadrupolar splitting is observed up to 25 D2O per lipid. Above this limit, the system goes from one phase (swollen lamellae) to two phases (swollen lamellae plus bulk water). In the swelling regime, i.e., between 10 and 25 D2O per lipid (hydration ranging from ca. 20 to 40%), the behavior of the observed quadrupolar splitting is accounted for by a two-site exchange theory. Water rapidly exchanges between motionally restricted and isotropic-like locations (membrane surface and swelling intermembrane space). Here, with (34) Douliez, J. P.; Bechinger, B.; Davis, J. H.; Dufourc, E. J. C-C bond order parameters from 2H and 13C solid-state NMR. J. Phys. Chem. 1996, 100, 17083-17086. (35) Douliez, J. P.; Le´onard, A.; Dufourc, E. J. Conformationnal approach of DMPC sn-1 versus sn-2 chains and membrane thickness: an approach to molecular protrusion by solid state 2H-NMR and neutron diffraction. J. Phys. Chem. 1996, 100, 18450-18457. (36) Douliez, J. P.; Ferrarini, A.; Dufourc, E. J. On the relationship between C-C and C-D bond order parameters and its use for studying the conformation of lipid acyl chains in biomembranes. J. Chem. Phys. 1998, 109, 2513-2518. (37) Bouglet, G.; Ligoure, C.; Bellocq, A. M.; Dufourc, E. J.; Mosser, G. Bending moduli of nonadsorbing-polymer-containing lyotropic lamellar phase: an experimental study. Phys. Rev. 1998, 57, 834-842. (38) Auguste, F.; Barois, P.; Fredon, L.; Clin, B.; Dufourc, E. J.; Bellocq, A. M. Flexibility of molecular films as determined by deuterium solidstate NMR. J. Phys. (Paris) 1994, 4, 2197-2214. (39) Auguste, F.; Douliez, J. P.; Bellocq, A. M.; Dufourc, E. J.; GulikKrzywicki, T. Evidence for multilamellar vesicles in the lamellar phase of an electrostatic lyotropic ternary system. A solid state 2H-NMR and freeze fracture electron microscopy. Langmuir 1997, 13, 666-672. (40) Faure, C.; Bonakdar, L.; Dufourc, E. J. Determination of DMPC hydration in the LR and Lβ′ phases by 2H solid-state NMR of D2O. FEBS Lett. 1997, 405, 263-266.
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the bicelles, one may invoke a similar behavior: water exchanging between the bicelle surface and the swelling interspace. Interestingly, using eq 4 (∆νobs ) nD∆νD/Ri) in Faure et al., which predicts the variation of the D2O quadrupolar splitting as a function of Ri, the water-tolipid molar ratio, one accounts for Figure 8, within the experimental error, by taking nD × ∆νD ) 10 × 1200 Hz, as for pure DMPC. This suggests that water bound to the bicelle membrane (for X ) 78%) has the same surface properties as those in DMPC. This also suggests that bicelles are subject to swelling for elevated water contents (up to 95%, i.e., for about 575 water molecules per lipid) as observed in pure lipid membranes for low hydration (up to 40%). This reinforces the deswelling hypothesis for better orientation in the presence of salts. This makes the bicelle a very interesting membrane system with a solvent exhibiting ordering properties in biologically relevant hydration conditions. Schematic Representation of the System. It may be interesting to give a pictorial representation of phases and phase changes that are encountered in our system. Although for some regions of the temperature-composition-hydration diagram this may be highly speculative due to the lack of experimental data, most of the longchain to short-chain lipid mixtures are basically encountered under the form of three main phases: micelles, bicelles, and lamellar phases. According to Israelashvili’s shape concept41 DCPC has a strong tendency to form structures of very high curvature, i.e., oil-in-water micelles. On the other hand DMPC forms bilayers that extend over long distances, several micrometers, that eventually close up to reduce edge energy. This is the well-known picture of multi- or uni-lamellar liposomes. So the phases that may be encountered in the diagram are compromises between these two opposite tendencies, high curvatures and planar structures. For elevated DCPC contents a pure mixed micellar phase is detected, the short-chain lipid imposing the curvature. At converse, the lamellar phase in encountered for low DCPC concentrations. For 6590% DMPC content, a micellar phase is detected below 20 °C. Here one may invoke that DMPC, even in high
concentrations, has not enough internal flexibility to compensate for the DCPC detergent-like effect. Mixed micelles are then detected. When the temperature above the gel-to-fluid phase transition of DMPC is increased, the acyl chains gain enough flexibility to accommodate both a high curvature and a planar structure. The DCPC compensates for the elevated edge energy that would otherwise be found for the contact of long acyl chains with water. This is the bilayer-micelle or bicellar phase. Upon further increase in temperature, a micellar phase coexisting with a lamellar phase is encountered. Preliminary centrifugation experiments indicate that there is indeed a physical phase separation above 40 °C, for DMPC concentration ranging between 65 and 90%.42 The micelle phase then observed is DCPC enriched whereas the lamellar phase contains more DMPC than the initial concentration mixture. This is also explained by the temperature-driven greater solubility of DCPC in water. For low temperatures, the short-chain lipids partition in an oil-like phase (DMPC) whereas for high temperatures they become more soluble. One must however warn the reader that even if the Israelashvili concept can account most of observables, it still represents a first-order description of our system. For instance, the stabilization of flat field orientable bicelles from mixed micelles is still not well understood.
(41) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of self-assembly of lipid bilayers and vesicles. Biochim. Biophys. Acta 1977, 470, 185-201.
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Conclusion 31P solid-state NMR has proven to be very powerful in determining the presence of bicelles and hence delineating the domain of existence where they self-orient. On the other hand, 2H NMR suggested that the bicelle core is more fluid than a pure long-chain lipid membrane and that all the water in the sample is ordered up to 95% hydration. It is also interesting to note that phosphorus31 NMR line shapes could be used to monitor the degree of bicelle orientation in the magnetic field.
Acknowledgment. J.H.D. thanks the NATO exchange program for funding part of this study.
(42) Arnold, et al. In preparation.
