INTRODUCTION

TOP ABSTRACT INTRODUCTION THEORY MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSION REFERENCES

The spontaneous orientation of phospholipid membranes in magnetic fields has often been considered problematic due to the resulting change in the solid-state nuclear magnetic resonance (NMR) spectral lineshapes (Bayerl and Bloom, 1990; Van Etcheld et al., 1982; Killian and de Kruijff, 1986). However, this phenomenon is now widely exploited since it has been shown to improve the spectral resolution (Sanders and Prestegard, 1990; Vold and Prosser, 1996). Phospholipids spontaneously form unilamellar and multilamellar vesicles, which are usually spherical in an aqueous medium (Helfrich, 1973; Pidgeon et al., 1987), but some membranes, depending on composition, concentration, temperature, and the method of preparation, tend to modify their shape in the presence of a magnetic field (Seelig et al., 1985; Speyer et al., 1987; Jansson et al., 1990; Qiu et al., 1993). When sterols (Brumm et al., 1992; Reinl et al., 1992) or proteins (Neugebauer et al., 1977; Van Echteld et al., 1982; Pott and Dufourc, 1995) are embedded in phospholipid membranes, the orientation may be enhanced or modified.

The origin of the magnetic alignment for a given molecule is its diamagnetic or paramagnetic anisotropy (Boroske and Helfrich, 1978). If a diamagnetic molecule is placed in a magnetic field, a magnetic moment proportional to the diamagnetic anisotropy is induced. Most of the time, the magnetic energy of one molecule is not sufficient to allow its alignment. However, when several molecules are packed together, there may be a reorientation of the aggregate relative to the field (Qiu et al., 1993). The diamagnetic anisotropy of an axially symmetric molecule, , is defined by the difference between the diamagnetic susceptibility parallel () and perpendicular () to the main axis of the molecule. For molecules with a negative , a perpendicular alignment will occur whereas those with a positive will orient parallel to the field. For example, in phospholipids, the hydrocarbon chains, which are considered to be the reference axis, have a  < 0 (Boroske and Helfrich, 1978), whereas glycerol ester carbonyls have a  > 0 (Maret and Dransfeld, 1985). Because glycerol carbonyls are about perpendicular to the phospholipid hydrocarbon chains (Sakurai et al., 1980), they both tend to orient the long axis of the lipid at 90° relative to the magnetic field. Peptide bonds also have a positive (Worcester, 1978). Because all peptide bonds contained in  or  helices are parallel to the helix axis, helical proteins will tend to orient parallel to a magnetic field (Neugebauer et al., 1977).

Proteins can also contain aromatic residues that are known to have large negative (Maret and Dransfeld, 1985). In particular, gramicidin A (GA) has 4 tryptophan residues in which the planes of the aromatic rings are approximately parallel to the helix axis (Ketchem et al., 1997), favoring a parallel orientation. In the same way, an aromatic molecule such as adriamycin (ADM) is expected, due to its negative , to orient its plane parallel relative to a magnetic field. In fact, it has been shown that the anthraquinone moieties of ADM molecules are closely stacked together when they are present at high concentration on the surface of a negatively charged phospholipid membrane (Goormaghtigh and Ruysschaert, 1984; De Wolf et al., 1991). Their magnetic susceptibilities are therefore added up, which makes possible the orientation of the membrane. The orientation of this membrane will depend on the angle formed by the ADM and the normal to the bilayer. If the ADM molecule is tilted by 39° (Goormaghtigh et al., 1987), the membrane would orient its normal parallel to the magnetic field.

The shape of phospholipidic membranes under high magnetic field is usually prolate ellipsoid (Helfrich, 1973; Speyer et al., 1987). These structures have been observed by electron microscopy (Brumm et al., 1992), ³¹P NMR (Pott and Dufourc, 1995; Brumm et al., 1992) and ²H NMR spectroscopies (Reinl et al., 1992; Schaefer et al., 1998). In addition, specific phospholipid mixtures are known to form bicelles, which are discoidal micelles formed when a long chain lipid (such as dimyristoylphosphatidylcholine (DMPC)) is mixed either with a short chain lipid (such as dihexanoylphosphatidylcholine (DHPC)) (Sanders and Schwonek, 1992; Vold and Prosser, 1996; Sanders and Prosser, 1998), a bile salt such as sodium glycocholate (Ram and Prestegard, 1988), or the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) (Sanders and Prestegard, 1990; Hare et al., 1995). In addition, it was recently observed that the addition of small amounts of paramagnetic ions to pure bicelles results in systems in which the director is oriented parallel to the magnetic field (Prosser et al., 1996). This phenomenon is due to paramagnetism and will not be considered here.

Solid-state ³¹P NMR spectroscopy is an ideal technique to follow the orientation of phospholipid membranes due to the presence of only one phosphorus atom in each phospholipid and the absence of phosphorus in most proteins. In addition, several interactions studied by ³¹P NMR are orientation dependent, such as the dipolar coupling and the chemical shift anisotropy (CSA) (Seelig, 1978; Smith and Ekiel, 1984). Spectral simulations have been done to extract the ratio of ellipsoid long/short axis from experimental ³¹P (Pott and Dufourc, 1995; Brumm et al., 1992) and ²H NMR spectra (Reinl et al., 1992; Schaefer et al., 1998). In contrast, even though ³¹P and ²H NMR spectroscopies have been applied to bicelles, no spectral simulation of such system has been done to our knowledge. However, the shape of the bicelles has been investigated by Chung and Prestegard (1993) based on field gradient studies and by Vold and Prosser (1996) based on the ratios of the deuteron splittings for DHPC and DMPC.

In the present paper, a method is proposed that uses the first spectral moment of ³¹P NMR spectra to obtain structural details on magnetically oriented phospholipid membranes. More specifically, it can be used to determine either the dimension of a bicelle or the ratio of an ellipsoid long/short axis. All these parameters are extracted from equations that are derived in the Theory section. Different systems are studied to present several of the possibilities mentioned above. First, DMPC forms ellipsoidal vesicles in which the phospholipids have a perpendicular orientation relative to the magnetic field. Moreover, a DMPC:DHPC mixture gives bicelles that align the bilayer normal perpendicular to the magnetic field. Finally, the lipids in membranes made of DMPC:DHPC and GA have a parallel orientation relative to the field, such as those in a complex made of a mixture of cardiolipin (CL) and ADM.

	THEORY

TOP ABSTRACT INTRODUCTION THEORY MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSION REFERENCES

In axially symmetric systems, ³¹P NMR chemical shifts are defined as

(1)

where  is the angle between the principal axis of the chemical shift tensor and the static magnetic field, and _iso is the isotropic chemical shift. To simplify this equation, the CSA parameter, , and the isotropic chemical shift are expressed in frequency units. Due to this chemical shift orientation dependence, both the spectral lineshape of a static sample and the weighted-average frequency of a sample with fast molecular motions can be related to the orientation distribution of the molecules in the sample. Therefore, if partial orientation occurs during an NMR experiment, a modification of either the lineshape or of the weighted-average frequency will also be present. Thus, information about the orientation distribution can be obtained from a detailed analysis of the spectral modification.

For static samples, a relationship between the spectral density, S(), and the angular distribution, P(), called the principle of differential conservation of the integral (Schmidt-Rohr and Spiess, 1994) is given by

(2)

That implies

(3)

In a randomly distributed sample, the angular distribution is easily calculated and the weighted-average frequency is equaled to the isotropic chemical shift, but, in lyotropic liquid crystals, partial orientation can occur due to the diamagnetic anisotropy. In this case, the orientational distribution has to be calculated. In the next section, two cases of partial orientation will be considered, an ellipsoid orientation distribution and a discoidal orientation distribution called bicelles.

Angular distribution and spectral lineshape

The angular distribution for ellipsoids has already been investigated by Pott and Dufourc (1995). Our approach is very similar to theirs but the following presentation is still necessary for a better understanding of that used for bicelles. Also, the description of the origin of the ellipsoidal angular distribution will help to define the effect of partial orientation on the first spectral moment.

Ellipsoids

(4)

View larger version (14K): [in this window] [in a new window]   FIGURE 1   Geometrical representations of (A) an ellipsoid, (B) a bicelle in the x'-y' plane, and (C) a bicelle in the (x'y')-z' plane.

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Oriented bicelles

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Order parameters S_dist and S₁

As discussed above, if there is a change in the shape of the orientation distribution, there is also a change in the weighted-average frequency. Rearranging Eq. 23, it is possible to define a distribution order parameter,

(27)

It should also be mentioned that the weighted-average frequency is exactly the same as a first moment calculation, i.e., M₁ = . Therefore, a calculation of the spectral moments could be a measure of the orientation. Obviously, this equation is only valid if the main axis of the distribution is along z. If the system main axis is tilted relative to the magnetic field axis, a new total order parameter can be introduced,

(28)

This new order parameter is denoted S₁ to emphasize that it comes experimentally from a first moment calculation. It is possible to solve these order parameter equations for many systems by considering their angular distributions. For a spherical vesicle, S₁ = 0. In the case of an ellipsoid, assuming that the main axis is along z, the order parameter S₁ is

(29A)

If r < 1,

(29B)

whereas, if r > 1,

(29C)

For bicelles, because their main axis can be oriented at different angles relative to the external magnetic field, it is more appropriate to define a distribution order parameter using the distribution of orientation and Eq. 22,

(30)

Then, the S₁ order parameter is determined from Eq. 28 using an S_tilt value of 1 for parallel bicelles and 0.5 for perpendicular bicelles. It is also possible to use any other S_tilt values for partially tilted bicelles.

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION THEORY MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSION REFERENCES

Material

DMPC, DHPC, and CL were obtained from Avanti Polar Lipids (Alabaster, AL) and used without further purification. GA and ADM (doxorubicin hydrochloride) were obtained from Fluka (Ronkonkoma, NY) and used without further purification.

Sample preparation

Aqueous dispersions of DMPC and CL were prepared in a 150 mM NaCl and 10 mM EDTA solution and adjusted to pH 6.5. Samples containing 20% (wt/wt) of lipids were then heated to ~50°C for 10 min, stirred on a vortex mixer, and cooled down at 0°C for 10 min. This cycle was repeated at least five times just before the analysis. The solution of ADM (0.01 g/mL) was prepared in 150 mM NaCl and 10 mM EDTA and adjusted to pH 6.5. The appropriate volume of the ADM solution was added to the CL dispersion to obtain a 2:1 CL:ADM molar ratio and then, five freeze-thaw cycles were applied to the system. Samples of 2.7:1 DMPC:DHPC molar ratio were prepared in a 0.1 M KCl buffer. Then samples containing 20% (wt/wt) of lipids were heated to ~35°C for 10 min, stirred on a vortex mixer, sonicated, and cooled down at 0°C for 10 min. This cycle was repeated at least five times just before the analysis. Samples of 10:4.3:1 DMPC:DHPC:GA molar ratio were prepared in trifluoroethanol. To obtain homogeneous peptide/lipid systems, the samples were incubated at 52°C for 1 h and shaken on a vortex mixer at least a few times during the incubation cycle. After the incubation, the organic solvent was evaporated with a nitrogen stream followed by high vacuum pumping overnight. The samples were then hydrated at 32% (wt/wt) with a HEPES buffer prepared at pH 7.0 and were submitted to several cycles of heating (52°C), vortex-mixer, sonication, and cooling (0°C).

NMR experiments

The ³¹P NMR spectra were acquired at 121.5 MHz on a Bruker ASX-300 (Bruker Canada Ltd., Milton, ON) operating at a ¹H frequency of 300.0 MHz. Experiments were carried out with a broadband/¹H dual frequency 4-mm probehead. The free induction decays (2 K data points) were recorded with a spin echo sequence (2000 or 4000 scans) with a 4 to 7 s repetition time and under conditions of proton decoupling. The ¹H 90° pulse length was typically 4.0 µs, corresponding to a rotating-frame frequency of about 63 kHz. The ³¹P 90° pulse length was 5 µs and the interpulse delay was set to 30 µs to avoid anisotropic T₂ effects on static spectra. The temperature was controlled to within ±0.5°C and the chemical shifts expressed in parts per millions (ppm) were referenced relative to the signal of phosphoric acid at 0 ppm. When not specified, a line broadening of 50 Hz was applied to the spectra. Magic angle spinning (MAS) ³¹P NMR spectra were obtained with a spinning speed of 6 kHz and a free induction decay of 4 K data points (1000 scans). No line broadening was applied to these spectra.

Simulations and calculations

Simulations and calculations were performed with the Grams 386 software (Galactic Industries Corp., Salem, NH) using the Array Basic programming language. The simulated spectra were broadened by convolution with a Gaussian function. The experimental spectral treatment was done using the UXNMR software from Bruker (Bruker Canada Ltd., Milton, ON).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION THEORY MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSION REFERENCES

Theoretical spectra

In this section, we will present the simulated ³¹P NMR spectra for partially oriented membranes, using the formalism developed in the Theory section. The simulated spectra will then be used to obtain the order parameters S₁ and S₂, respectively related to the orientation and the dynamics of membranes.

Figure 1 shows two types of partially oriented membranes, an ellipsoidal vesicle in A and two views of a bicelle in B and C. If partial orientation occurs in model membranes, they will most likely adopt an ellipsoidal structure in which a will be smaller than c due to the sign of the lipid diamagnetic anisotropy (Boroske and Helfrich, 1978). In this kind of system, the majority of the phospholipids will be oriented with their main axis, , at 90° relative to the surface. Such an ellipsoidal orientation implies either very large unilamellar or multilamellar vesicles because phospholipids in an ellipsoid with a very short semi-axis a will experience a fast angular diffusion that would result in chemical shifts characteristic of a hexagonal phase rather than the ones characteristic of lamellar phases (Seelig, 1978).

Figure 2 shows the simulated spectra of ellipsoidal structures for r ranging from 5 to 0.2. At a ratio of 5, the phospholipids are almost all oriented at 90° relative to the magnetic field. The ratio of 1 corresponds to a spherical vesicle, and, at a ratio of 0.2, the majority of the phospholipids is oriented parallel to the magnetic field. The surprising point of these simulated spectra is that a prolate ellipsoid gives a very small distribution of frequencies in comparison with the broad distribution occurring for an oblate shape. This can be seen in Fig. 2 by comparing the spectra with r = 2.5 and 0.4. 

View larger version (20K): [in this window] [in a new window]   FIGURE 2   31P NMR spectral simulations of ellipsoidal vesicles as a function of r with  = 25 ppm, iso = 0 ppm and a Gaussian broadening (FWHH = 1 ppm).

The second case of partial orientation is the bicelle, which is usually made of a long acyl chain phospholipid that has been assumed to be on the two circular planes, and of a short acyl chain phospholipid that has been assumed to be on the rim of the bicelle (Sanders and Schwonek, 1992; Vold and Prosser, 1996). Bicelles made of only two circular planes have also been proposed for systems composed of CHAPSO and DMPC (Sanders and Prestegard, 1990; Sanders et al., 1994). In the present study, the bicelles are modeled by the combination of the outer part of a torus and of two circular planes (Fig. 1, B and C). Simulations will be made using two different models. In the first model, we will consider a constant molecular surface area, a constant composition of phospholipids over the whole surface of the bicelle and a negligible contribution of the lipid lateral diffusion. In the second model, we will consider a rapid axial rotation of the bicelles around their main axis in addition to a rapid lateral diffusion of the lipids. In these two models, the bicelles are considered to be made of a single bilayer in which the thickness is the double of the radius of the edge, as illustrated in Fig. 1 C. The axis system of the bicelles is denoted (x', y', z') to account for the difference with the laboratory frame (x, y, z). Two different orientations of the main axis of the bicelles are modeled, a bicelle with its z' axis along z and a bicelle with its x' axis along z.

Figure 3 A reports the simulation of ³¹P NMR spectra of static parallel bicelles in which the effect of the orientation can be easily seen as an increasing spectral component at . An important point to note is that a highly oriented system, e.g., with an r value of 0.5 corresponding to a bicelle thickness of 50 Å and to a bicelle total diameter of 200 Å, gives a broad spectrum if the main axis of the bicelle is oriented parallel to the magnetic field. However, because the broad component is due to the rim of the bicelle, static parallel bicelles should give sharp NMR peaks for molecules exhibiting fast axially symmetric motions inserted into their flat sections, such as membrane proteins. Finally, the spectral difference between parallel bicelles and oblate spheroids allows a discrimination of the two types of organization, as discussed in a later section.

View larger version (31K): [in this window] [in a new window]   FIGURE 3   31P NMR spectral simulations of bicelles as a function of r with  = 25 ppm, iso = 0 ppm and a Gaussian broadening (FWHH = 3 ppm). (A) Static parallel bicelles. (B) Parallel bicelles with axial tumbling and lateral diffusion. (C) Perpendicular bicelles with axial tumbling and lateral diffusion.

Figure 3, B and C, shows simulated parallel and perpendicular bicelle spectra in which tumbling and lateral diffusion have been considered. In this model, we supposed that the phospholipids that constitute the torus do not diffuse in the planes and vice-versa. This assumption has been made because experimental bicelles are constituted of two types of phospholipids that are believed to be laterally phase separated. The relative proportion of the two peaks observed in these spectra varies with the r shape parameter. This relationship is defined by Eq. 22 and is plotted in Fig. 4 A. Therefore, from an experimental spectrum, it is possible to determine the size of the bicelle only by integrating the two peaks. The frequency of the peak from the torus part can also be related to the r shape parameter by

(31)

An S_dist order parameter can be calculated from this weighted-average frequency using Eq. 27. This relationship, plotted in Fig. 4 A, could also be a measure of the r shape parameter. A value of 0.25 for the center of gravity of the torus section of bicelles relative to the bilayer normal has already been proposed in the literature (Vold and Prosser, 1996) for ²H NMR spectra.

View larger version (24K): [in this window] [in a new window]   FIGURE 4   (A) Relative proportion, , and Sdist for the lipids in the torus part of the bicelles. (B) S1 order parameter for ellipsoidal vesicles, parallel bicelles, and perpendicular bicelles as a function of the shape parameter r.

As discussed before, in oriented systems, it is possible to define an order parameter from the first spectral moment measured experimentally, which will be a measure of the orientation level. A system with a value of S₁ close to 1 (0.5) indicates that all the molecules are oriented with their main axis parallel (perpendicular) to the magnetic field. Figure 4 B reports the variation of S₁ with the shape parameter r assuming that the main axis of the oriented system is along z for ellipsoids and either along or perpendicular to z for bicelles.

A relative order parameter can also be defined, namely the ratio of the spectral widths of an experimental spectrum to that of a reference spectrum,

(32)

This order parameter, denoted S₂ to emphasize that it is related to the second spectral moment, can vary from 1 for a system in which the dynamics are the same as those in a reference system to 0 if the system becomes totally isotropic. Several reference systems can be chosen, such as the CSA of DMPC multilamellar vesicles when investigating bicelles made of DMPC and DHPC. This order parameter combines the effects of two other order parameters, a first one that has already been introduced in the literature as S_bilayers (Sanders and Schwonek, 1992), which denotes the presence of local motional averaging of the CSA, and a second that we have previously named S_tilt and that some authors have introduced as S_system (Sanders et al., 1994), which relates the orientation of the main axis of the system to the magnetic field axis. However, in the present study, the main axis of each system is considered to be oriented either parallel or perpendicular to z, which implies that a change in S₂ is considered as a change in the dynamic properties (lateral diffusion, wobbling, tumbling) of the lipids rather than an orientational change of the main axis of the oriented system (such as a tilt of the bicelles).

Experimental systems

To validate the spectral simulations and the use of the order parameters presented in the previous section, several partially oriented lipid systems have been investigated. More specifically, we have first investigated the partial orientation of pure DMPC multilamellar vesicles as a function of temperature and of hydration level. This system is known to be partially deformed in high magnetic fields, and the results of the present study will show that the partial orientation can be quantitatively defined by the order parameter S₁. A similar approach will then be applied to DMPC:DHPC bicelles, a lipid system known to orient with the lipids oriented at 90° relative to the magnetic field. Finally, we will investigate two cases of partial parallel lipid orientation, namely the parallel orientation of DMPC:DHPC membranes in the presence of GA and the partial orientation of CL membranes in the presence of ADM.

Pure DMPC

View larger version (15K): [in this window] [in a new window]   FIGURE 5   (A) Experimental 31P NMR spectra. (B) r Shape parameter for DMPC as a function of temperature and concentration (wt/wt %).

Bicelles

View larger version (21K): [in this window] [in a new window]   FIGURE 6   (A) Experimental 31P NMR spectra. (B) S1 and S2 order parameters and (C) S1t order parameter and t for DMPC:DHPC (2.7:1 molar ratio) as a function of temperature.

(33)

Parallel lipid orientation

View larger version (21K): [in this window] [in a new window]   FIGURE 7   (A) Experimental 31P NMR spectra and (B) S1 and r for DMPC:DHPC:GA (10:4.3:1 molar ratio) as a function of temperature and time. The spectra have been recorded every four hours at the following temperatures: (a) 25°C, (b) 30°C, (c) 35°C, (d) 40°C, (e) 45°C, (f to l) 50°C.

Parallel lipid orientation without DHPC? the CL:ADM complex

View larger version (20K): [in this window] [in a new window]   FIGURE 8   (A) Experimental 31P NMR spectra and (B) S1 as a function of temperature for the CL:ADM (2:1 molar ratio) system. A line broadening of 150 Hz was applied to these spectra.

Perspectives

The concept of magnetic orientation is not really new. However, a lack of methods to evaluate this phenomenon is evident in this area of research. Some authors (Forrest and Reeves, 1981) proposed order parameters to evaluate the extent of magnetic orientation, but these parameters are not really appropriate. More specifically, the measurement of the orientation via an order parameter such as S₂, as described above, is interesting but not complete. This order parameter could be indicative if a tilt of the bicelles or ellipsoids relative to the magnetic field axis is assumed. However, in general, it is more appropriate to suppose that the main axis of the deformed membrane is along or perpendicular to the magnetic field. In these cases, such order parameters will provide interesting information about the dynamics in the system. Therefore, the definition of another order parameter is really important. In the previous section, we proposed a new order parameter, S₁, derived from the measurement of the first spectral moment.

Spectral moments are well defined in the literature and are very easy to measure (Abragam, 1961). Therefore, an orientation parameter derived from this type of measurement is suitable. In fact, a change in the spectral lineshape due to partial orientation will affect the spectral moments. In principle, it could be possible to obtain orientation-dependent measurements from each of the spectral moments (M₁, M₂, M₃, M₄, ...). However, the moments higher than the first one are very dependent on the spectral line broadening and only M₁ is not affected by symmetrical broadenings, such as Lorentzian or Gaussian broadenings. Therefore, only the use of M₁ is possible, even if M₁ is dependent on both the CSA and the isotropic chemical shift. With an evaluation of  on a well-defined static spectrum and of _iso from a MAS spectrum, a really precise evaluation of S₁ is possible.

Because of the increasing use of both parallel and perpendicular bicelles as model membrane systems, the characterization of their shape and size is important. A model has recently been proposed based on ²H NMR measurements and on the calculation of the relative areas of the planes and edges of the bicelles (Vold and Prosser, 1996). There are also extensive studies of the position of DHPC on the edges of the bicelles by ²H and ³¹P NMR spectroscopy (Sanders and Schwonek, 1992; Vold and Prosser, 1996). However, there are no simulated spectra of such bicelles in the literature, and therefore, an attempt to simulate both the perpendicular and parallel orientation of bicelles seems valuable.

The experimental spectra obtained for bicelles with their main axes oriented parallel to the magnetic field are in agreement with simulated spectra, suggesting the validity of this model. For perpendicular bicelles made of DMPC and DHPC, the presence of a second peak with a frequency different from _iso is a convincing proof of the validity of the bicelle model. In addition, both the relative intensity of the two peaks and the S₁ calculation give the same r shape parameter, which corresponds to the proposed diameter of the bicelles. Finally, the values of S₁ obtained for the phospholipids in the edges of the bicelles suggest that there is a phase separation between the lipids in the planes and the torus of the bicelles and that there is a fast exchange process between them. These results from ³¹P NMR are in agreement with those obtained from ²H NMR measurements (Vold and Prosser, 1996).

	CONCLUSION

TOP ABSTRACT INTRODUCTION THEORY MATERIAL AND METHODS RESULTS AND DISCUSSION CONCLUSION REFERENCES

A new order parameter, S₁, has been proposed in the present study to obtain quantitative information about the orientation of phospholipidic systems. This new tool can be helpful both to investigate the shape of lipid membranes and to study the effect of several parameters (temperature, hydration, addition of proteins or drugs, etc.) on the orientation of phospholipidic systems. A second order parameter, S₂, can provide complementary information about the dynamics in an oriented system. ³¹P NMR spectra have been simulated for both ellipsoid and bicellar systems in which the lipids are oriented either parallel or perpendicular to the external magnetic field. In addition, the S₁ and S₂ order parameters have been determined from the experimental spectra obtained for several systems.

More specifically, the general influence of temperature and concentration on orientation has been clearly demonstrated for pure DMPC multilamellar vesicles. In addition, the orientation and shape of bicelles made of DMPC:DHPC, in which the lipids are oriented perpendicular to the magnetic field, has been determined as a function of temperature. We have also shown in this study that the addition of GA to the DMPC:DHPC system induces an orientation of the lipids parallel to the magnetic field that seems to favor the formation of bicelles. Adriamycin also induces an orientation of the lipids at 0° relative to the magnetic field in CL bilayers, even in the absence of the short chain lipid DHPC. This indicates that a partial positive ordering can be obtained in lipid systems without the addition of paramagnetic ions and can be well characterized by the order parameter S₁ derived from the first spectral moment.