INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Monoglycerides are amphiphatic neutral lipid molecules in which a hydrophobic fatty acid is attached at the sn-1(3) position of a hydrophilic glycerol backbone via an ester bond. Despite their relatively simple chemical structure, monoglycerides can form various phases found in membrane phospholipid/water systems, namely the coagel or lamellar crystalline (L_c) phase, the lamellar gel (L) phase, the lamellar liquid-crystalline (L) phase, bicontinuous cubic phases of different symmetry, the inverted hexagonal (H_II) phase, and the inverted micellar (L₂) phase. The lipid organization in monoglyceride-water systems is strongly dependent on the chemical structure of the monoglyceride and on environmental conditions such as temperature, water, and salt content.

The ability of monoglyceride/water systems to form different structures offers many interesting opportunities for studies on membrane structure and function, as well as for industrial applications. Being a natural emulsifier, monoglycerides are widely used in the food industry (Krog, 1990). During fat digestion the enzymatic hydrolysis of triglycerides leads to accumulation of a large amount of monoglycerides, which may result in formation of a bicontinuous cubic phase. Such a cubic phase could be a suitable matrix for interfacial enzymatic processes and for transport of lipid molecules between the fat droplets and mixed micelles of monoglycerides/fatty acids and bile acids (Lindblom and Rilfors, 1989). The monoglyceride cubic phase has also been successfully used as a matrix for crystallization of membrane proteins (Landau and Rosenbusch, 1996; Rummel et al., 1998; Kolbe et al., 2000).

In fat digestion and in industrial applications, monoglycerides are always present as mixtures with other lipids and proteins. The same is the case for crystallization of membrane proteins from the cubic phase of monooleoylglycerol. This phase is enriched by detergents, which are used for the solubilization of hydrophobic proteins. Potentially, additives introduced into monoglycerides can significantly change the properties of the system. For example, it was hypothesized that in the cubic phase of monoolein, protein crystals grow at the locally formed L phase, which is stabilized by detergents (Ai and Caffrey, 2000). For these reasons, mixed monoglyceride systems are of utmost importance for both biochemical research and industrial application. Although the polymorphic behavior of monoglyceride-water systems has been studied for many years (Lutton, 1965; Krog and Larson, 1968; Larsson and Quinn, 1994; Briggs and Caffrey, 1994; Qiu and Caffrey, 2000) little knowledge is as yet available on the properties of mixed monoglyceride systems. This applies in particular to systems containing proteins. Proteins are present in all systems mentioned above, and, therefore, the interaction between monoglycerides and proteins may play an important role in structural organization and function of these systems.

In our previous papers (Leenhouts et al., 1997; Boots et al., 1999, 2001) we reported on the interaction of -lactoglobulin and lysozyme with monoglyceride monolayers and vesicles. In this paper, we present results on the interaction of -lactoglobulin and lysozyme with a monostearoylglycerol/dicetylphosphate two-component mixture in different phase states. The negatively charged dicetylphosphate (DCP) (Fig. 1) was included because we observed previously (Leenhouts et al., 1997; Boots et al., 1999, 2001) that low concentrations of DCP facilitate insertion of proteins into monoglyceride monolayers. Lysozyme and -lactoglobulin have a similar size, 14.3-kDa versus 18.3-kDa, and number of positively charged amino acids, 17 versus 18. However, lysozyme contains far less negatively charged amino acids, 9 versus 26, giving it an isoelectric point of 9.3 compared with 5.2 for -lactoglobulin. Therefore, lysozyme is positively charged and -lactoglobulin is negatively charged at neutral pH. The questions, which we are addressing in this study, are 1) how the presence of DCP can affect the lipid organization and phase properties of the MSG/water system, and 2) how the negatively charged -lactoglobulin and the positively charged lysozyme interact with this monoglyceride system.

View larger version (9K): [in this window] [in a new window]   FIGURE 1   [2H35]-Monostearoyl-rac-glycerol ([2H35]-MSG) and DCP.

The lipid organization and phase behavior were studied by means of solid-state ²H and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Deuterium-labeled 1-[²H₃₅]-stearoyl-rac-glycerol ([²H₃₅]-MSG) (Fig. 1) with a fully deuterated acyl chain was used as a selective probe for ²H NMR. The behavior of DCP was followed by ³¹P NMR. In this approach, the behavior of individual molecular components can be monitored in the same sample, which is important for monoglyceride/water systems known to form long-living metastable phases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials

Bovine -lactoglobulin (a mixture of genetic variants A and B) and DCP were obtained from Sigma Chemical Company (St. Louis, MO). Lysozyme was obtained from Boehringer (Mannheim, Germany). Fully deuterated stearic acid-D₃₅, 13-[²H₂]-palmitic acid, and deuterium-depleted water were obtained from Cambridge Isotope Laboratories (Cambridge, MA). 1,2-Dipalmitoyl-sn-glycerophosphocholine was obtained from Avanti Polar Lipids (Alabaster, AL). 1-[²H₃₅]-Monostearoyl-rac-glycerol with a fully deuterated acyl chain was synthesized according to the procedure described previously (Chupin et al., 2001).

Sample preparation

Samples were prepared by mixing known amounts of [²H₃₅]-MSG and DCP stock solutions in CHCl₃/MeOH (3:1). The solvents were evaporated and then residual solvent was removed under high vacuum for at least 4 h. The lipids were subsequently hydrated by adding buffer and heating the samples at 65°C for at least 5 min and cooling to room temperature. After three cycles of heating and cooling, the samples were used for NMR experiments. The samples consisted of 50-70 µmol lipid (10% by weight) hydrated in buffer. Samples containing proteins were obtained by hydration of a dry lipid powder with a solution of -lactoglobulin or lysozyme in buffer without or with 100 mM sodium chloride. A molar lipid-to-protein ratio of 100 was used to achieve saturation in protein binding (Boots et al., 1999, 2001). All buffers were prepared with deuterium-depleted water: 20mM Tris, pH 7, with or without 100 mM NaCl. Potassium palmitate sample was prepared according to Davis and Jeffrey (1977) and contained 10% (mol) of DCP and 10% (mol) of 13-[²H₂]-palmitate.

NMR measurements

All NMR spectra were recorded on a Bruker MSL 300 spectrometer (Bruker Karlsruhe, Germany). Using a high-power 7.5-mm broadband probe, 46.1 MHz ²H NMR spectra were obtained, as described previously (Chupin et al., 2001). A quadrupolar echo technique (Davis et al., 1976) with a 3-µs /2-pulse and a 40-µs  delay was used. 121.1 MHz ³¹P NMR spectra were recorded using a high-resolution 10 mm broadband probe. A Cyclops sequence with broadband, gated proton decoupling was used. The recycling delay was 1 s and the /4-pulse width was 8 µs. Typically, 15 000 free induction decays for the L phase and ~1000 for the L and L₂ phases were accumulated. In the L_c phase, ³¹P NMR spectra were obtained using a 5-mm probe. A spin echo technique with a 6-µs /2-pulse, a 30-µs  delay, and 8-s recycling delay was used. An exponential multiplication with a line-broadening factor of 300 Hz for the L_c and L phases, and 30 Hz for the L and L₂ phases was used before performing the Fourier transformation. All ²H NMR spectra were symmetrized. Chemical shifts in ³¹P NMR spectra were measured relative to the isotropic signal.

Theory and application of ²H NMR (Davis, 1983; Seelig and Macdonald, 1987; Smith, 1989) and ³¹P NMR (Seelig, 1978; Smith and Ekiel, 1984) spectroscopy in lipid systems is described in the literature. ³¹P NMR spectra of DCP in the L phase were simulated using the Lorentzian broadening.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The phase behavior and lipid organization of a [²H₃₅]-MSG:DCP mixture and its interaction with -lactoglobulin and lysozyme were investigated in excess water by using ²H and ³¹P NMR. The [²H₃₅]-MSG:DCP mixture in a molar ratio of 9 to 1 was chosen based on our previous observations that even low concentrations of DCP facilitate interaction of -lactoglobulin and lysozyme with monoglycerides (Leenhouts et al., 1997; Boots et al., 1999, 2001). We previously demonstrated that ²H NMR spectroscopy on deuterated acyl chain monoglycerides is a convenient technique to monitor phase transitions in monoglyceride-water systems (Chupin et al., 2001) because all different phases give rise to characteristic features in the ²H NMR spectra. Similarly, ³¹P NMR should allow for following the behavior of DCP. However, the line shape of ³¹P NMR spectra of DCP in different phases is not known. Therefore, we first characterized DCP in different phases by ³¹P NMR.

³¹P NMR spectra of DCP in different phases

Although ³¹P NMR spectra of DCP in different phases have never been published, it is easy to predict the line shape for DCP in L₂ phases as well as in the L_c phase. In cubic or other isotropic phases, the fast isotropic motion of lipid molecules will result in a narrow symmetric signal in NMR spectra independent of the structure of these molecules (Lindblom and Rilfors, 1989). With respect to the coagel, it was shown that ³¹P NMR spectra of crystalline phosphates exhibit a broad powder type pattern with three components of the chemical shielding tensor. Principal tensor values of ~₁₁ = 80, ₂₂ = 20, and ₃₃ = 110 are almost identical for different diester phosphates, which is characteristic for immobilized phosphate groups (Seelig, 1978). Indeed, the ³¹P NMR spectrum of DCP in the crystalline bilayer exhibits similar features (Fig. 2 A) as the crystalline powder of phospholipids (Fig. 2 D).

View larger version (12K): [in this window] [in a new window]   FIGURE 2   31P NMR spectra of DCP and dipalmitoylphosphatidylcholine (DPPC) in different phases. (A) DCP in the Lc phase of potassium palmitate at 20°C (dry powder). (B) DCP in the L phase of potassium palmitate at 25°C. (C) DCP in the L phase of potassium palmitate at 50°C. (D) DPPC in the Lc phase at 20°C (dry powder). (E) DPPC in the L phase at 25°C. (F) DPPC in the L phase at 50°C.

However, the line shape of ³¹P NMR spectra of DCP or structurally related compounds in gel and liquid-crystalline bilayers is not known. As DCP may not form such phases on its own, the experimental spectra of DCP in the L and L phases were obtained by using potassium palmitate as a bilayer matrix (Davis and Jeffrey, 1977) that is transparent for ³¹P NMR. The phase state of the matrix was first checked by ²H NMR using [13-²H₂]-palmitate as a probe. ²H NMR demonstrated that the presence of a small amount of DCP does not affect the bilayer organization of potassium palmitate (not shown). ³¹P NMR spectra of a DCP:potassium palmitate mixture recorded below and above the L-to-L phase transition are shown (Fig. 2, B and C) and compared with the spectra of dipalmitoylphosphatidylcholine in the L and L phase (Fig. 2, E and F).

³¹P NMR spectra of DCP both in the L and L phases significantly differ from the spectra of phospholipids in the corresponding phases. In the L phase, the ³¹P NMR spectrum of DCP exhibits a line shape, which is characteristic of a partially immobilized phosphate group (Fig. 2 B). The spectrum of DCP in the L phase clearly has a nonaxial symmetry indicating that the phosphate group of DCP does not undergo fast rotation. Three components of the chemical shielding tensor are resolved with values of ~₁₁ = 51, ₂₂ = 10, and ₃₃ = 62 ppm at 25°C. These values are much smaller than those obtained for the L_c phase, indicating that some motions occur in the L phase. In contrast to DCP, ³¹P NMR spectra of phospholipids exhibit a line shape close to an axial symmetric pattern with parallel and perpendicular components (Fig. 2 E).

In the L phase, the ³¹P NMR spectrum of DCP has a high-field peak and low-field shoulder (Fig. 2 C) similar to phospholipids in liquid-crystalline bilayers (Fig. 2 F). However, the chemical shift anisotropy (CSA) of the DCP signal is ~15 ppm, which is much smaller than the values of ~40 ppm for most phospholipids.

The difference in line shape and CSA of DCP as compared with phospholipids can be explained by the chemical structure of DCP as will be discussed below.

Effect of DCP on the phase behavior of [²H₃₅]-MSG

In our previous paper we reported on ²H NMR spectra of [²H₃₅]-MSG in different phases (Chupin et al., 2001). As a control, these spectra are shown in Fig. 3, A-D. A pictorial description of the different monoglyceride phases is shown on the right panel. In the L_c phase of [²H₃₅]-MSG at 20°C, the spectrum is characteristic for immobilized acyl chains and consists of two well defined powder patterns, one for the methylene groups with a quadrupolar splitting (_Q)of 120 kHz and another for the methyl groups with a splitting of 35 kHz (Fig. 3 A). In the L phase at 40°C, the spectrum of [²H₃₅]-MSG again shows two resolved signals, but now with reduced _Q of ~50 kHz and 11 kHz, indicating partial motional averaging (Fig. 3 B). The L phase of [²H₃₅]-MSG is metastable and gradually transforms into the coagel during several hours at 20°C (not shown). The ²H NMR spectrum in the L phase of [²H₃₅]-MSG consists of a number of resolved resonances from different labeled sites along the acyl chain (Fig. 3 C). In the cubic phase (the cartoon shows the Im3m phase proposed for MSG (Lindblom et al., 1979)), the ²H NMR spectrum of [²H₃₅]-MSG shows a narrow signal originating from isotropically moving lipid molecules (Fig. 3 D).

View larger version (24K): [in this window] [in a new window]   FIGURE 3   2H NMR spectra (left panel) representing the different phases of [2H35]-MSG, as schematically depicted (right panel). (A) Lc phase; (B) L phase; (C) L phase; and (D) Cubic phase.

The presence of DCP significantly changes the phase behavior of the monoglyceride. This is shown by the ²H NMR and ³¹P NMR spectra of [²H₃₅]-MSG:DCP, 9:1 (mol/mol), at different temperatures (Fig. 4, A-F). Once heated above the Kraft point, the [²H₃₅]-MSG:DCP mixture forms a long-living L phase even at low temperatures, as can be concluded from the ²H NMR spectrum with _Q of ~50 and 11 kHz at 20°C consistent with the presence of a L phase (Fig. 4 A). The ³¹P NMR spectrum of this mixture (Fig. 4 D) shows a line shape with reduced anisotropy, which is a characteristic of the partially immobolized phosphate group of DCP in the L phase (cf. Fig. 2 B). In contrast to pure [²H₃₅]-MSG dispersions, the L phase of MSG:DCP is stable during months at room temperature and transforms into the coagel very slowly (not shown). Upon heating the L phase of [²H₃₅]-MSG:DCP, the _Q in the ²H NMR spectra and the total anisotropy in the ³¹P NMR spectra are reduced (Fig. 4, B and E), but no L phase is formed. Instead, upon further heating, the L phase of [²H₃₅]-MSG:DCP transforms directly into an L₂ phase as indicated by the narrow symmetric peaks in the ²H and ³¹P NMR spectra (Fig. 4, C and F). The L-to-L₂ phase transition in [²H₃₅]-MSG:DCP was found at 56°C for both lipid molecules (not shown), which is approximately the same as the L-to-L phase transition temperature of 55°C of pure [²H₃₅]-MSG indicating that no significant phase separation takes place. This was also confirmed by DSC data (not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 4   2H NMR (A-C) and 31P NMR (D-F) spectra of [2H35]-MSG:DCP, 9:1 (mol/mol) at different temperatures, as indicated in the figure.

Interaction of -lactoglobulin and lysozyme with [²H₃₅]-MSG:DCP

To investigate the possible effects of -lactoglobulin and lysozyme, the phase behavior of MSG:DCP mixtures was investigated in the presence of these proteins. At pH 7, the lipid bilayer surface is negatively charged because of the presence of DCP whereas lysozyme has a positive net charge and -lactoglobulin is negatively charged. Buffers with low and high ionic strength were used to modulate the electrostatic interactions between protein and lipid surface. NMR spectra of the lipid-protein mixtures were recorded in the temperature range from 20°C until the temperature of denaturation of -lactoglobulin and lysozyme, 70° and 65°C, respectively (Ericsson et al., 1983; Iametti et al., 1996).

Fig. 5 shows the ²H and ³¹P NMR spectra of a [²H₃₅]-MSG:DCP mixture which was hydrated with a solution of -lactoglobulin. At low temperature, both ²H and ³¹P NMR spectra (Fig. 5, A and D) exhibit a line shape which is representative of the L phase of the pure lipid system (cf. Fig. 4, A and D). The L phase of the [²H₃₅]-MSG:DCP:-lactoglobulin mixture slowly transforms into the coagel during months at 20°C with approximately the same rate as a [²H₃₅]-MSG:DCP mixture (data not shown). Apparently, the presence of protein does not affect the stability of the L phase. Upon heating, the L phase of the [²H₃₅]-MSG:DCP:-lactoglobulin mixture transforms into the L phase (Fig. 5, B and E). Strikingly, the presence of -lactoglobulin thus completely neutralizes the destabilizing effect of DCP on the L phase of [²H₃₅]-MSG (cf. Fig. 4, B and E and Fig. 5, B and E), indicating that the protein interacts with [²H₃₅]-MSG:DCP bilayers. The protein even stabilizes the L phase of [²H₃₅]-MSG:DCP, which is now more stable as compared with the pure [²H₃₅]-MSG. No isotropic signal was observed up to a temperature of 70°C at low ionic strength, whereas the pure [²H₃₅]-MSG transforms into the cubic phase at 67°C (Chupin et al., 2001). At high ionic strength, this effect is decreased and the L phase of [²H₃₅]-MSG:DCP transforms into the L₂ phase at a temperature of 65°C, indicating that electrostatic interactions are involved. Control experiments demonstrated that -lactoglobulin does not significantly affect the phase behavior of pure [²H₃₅]-MSG (not shown), indicating that the presence of DCP is of importance for the interaction of this negatively charged protein with the negatively charged lipid surface.

View larger version (16K): [in this window] [in a new window]   FIGURE 5   2H NMR (A-C) and 31P NMR (D-F) spectra of [2H35]-MSG:DCP, 9:1 (mol/mol) in the presence of -lactoglobulin at different temperatures. Lipid:protein ratio, 100:1 (mol/mol); pH 7; 0 mM NaCl.

The positively charged lysozyme has a similar effect on the phase behavior of [²H₃₅]-MSG:DCP mixtures as -lactoglobulin (not shown). Lysozyme stabilizes the L phase at the expense of the L₂ phase up to 65°C, the temperature of its denaturation. The presence of lysozyme does not accelerate transformation of the metastable gel into the stable coagel, again similar to the effect of -lactoglobulin. In the presence of either protein, the transition into the L phase occurs at the same temperature for both [²H₃₅]-MSG and DCP (not shown), indicating that there is no phase separation of lipids involved.

Interestingly, the line shape of the ²H NMR spectra of [²H₃₅]-MSG in the L phase (Fig. 6 A) is clearly different from that of the mixture of [²H₃₅]-MSG:DCP:protein (Fig. 6 B). In the presence of protein, the spectrum exhibits lower resolution and decreased intensity of the signal with the largest _Q. This signal is a superposition of the unresolved resonances of the acyl chain methylenes located near the monoglyceride headgroup. In the presence of protein the order in this region is decreased, indicative of lipid-protein interactions. There are two possible explanations of this effect. The first is that insertion of protein molecules into the bilayer decreases the resolution of the spectrum (Fig. 6 B). This effect could be attributable to the destruction of the uniform orientation of the acyl chains and/or slowing down of the acyl chain motion, which reduces the deuteron T₂ relaxation time. The second is that the resolution in the spectrum of [²H₃₅]-MSG is higher because of the magnetic orientation of the lipid bilayers (Chupin et al., 2001) and the proteins destroy this orientation in [²H₃₅]-MSG:DCP mixture. Taking our data obtained on MSG:DCP monolayers (Boots et al., 1999, 2001) into account, the first explanation seems more likely.

View larger version (11K): [in this window] [in a new window]   FIGURE 6   2H NMR spectra of [2H35]-MSG (A) and [2H35]-MSG:DCP, 9:1 (mol/mol) in the presence of lysozyme (B) in the L phase at 60°C. Lipid:protein ratio, 100:1 (mol/mol); pH 7; 0 mM NaCl.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

³¹P NMR spectra of DCP in different lamellar phases

The line shape of ³¹P NMR spectra is determined by the values of the principal elements of the shielding tensor, its orientation, and averaging because of molecular and intramolecular motions. The principal elements of the shielding tensor of different phosphodiesters are almost identical (Seelig, 1978). Their values are of ₁₁ = 80, ₂₂ = 20, and ₃₃ = 110 ppm. The orientation of the chemical shielding tensor with respect to the molecular frame is known for barium diethylphosphate (Herzfeld et al., 1978), which is structurally related to DCP. The ₁₁ element is approximately perpendicular to the O(3)-P-O(4) angle and the ₂₂ element approximately bisects the O(3)-P-O(4) angle, where O(3) and O(4) are nonesterified oxygen atoms. The problem then is how to relate the orientation of the chemical shielding tensor with respect to the molecular frame of DCP in a bilayer. Fortunately, a DCP molecule has a symmetric shape. Both alkyl chains attached to the phosphate group are chemically equivalent, as are the nonesterified oxygen atoms. Therefore, it is reasonable that both chains are embedded into the bilayer in the same way, whereas both charged nonesterified oxygens are exposed into the water phase again in the same way. In this case, the direction of the ₂₂ element coincides with the bilayer normal and the elements ₁₁ and ₃₃ are parallel to the bilayer plane (Fig. 7 A).

View larger version (9K): [in this window] [in a new window]   FIGURE 7   Different orientation of the diester phosphate group of DCP (A) and phosphatidylcholine (B) in a bilayer with respect to the bilayer normal, n. The orientation of the shielding tensor is shown for DCP (A). Arrows indicate possible intramolecular rotations of the phosphatidylcholine headgroup, which can reduce the CSA (B). Simulated (a) and experimental (b) 31P NMR spectra are shown for DCP in the L lamellar phase (C). The simulated spectrum was obtained by using  = 15 ppm and 200-Hz line broadening.

In the L_c phase, the ³¹P NMR spectrum of DCP exhibits a line shape with three components of the shielding tensor similar to other crystalline diester phosphates (Fig. 2 A). Qualitatively, we observed the same line shape in the L phase of DCP (Fig. 2 B). However, the total CSA of DCP is significantly reduced in the L phase compared with the L_c phase, as indicated by the values of ₁₁ = 51, ₂₂ = 10, and ₃₃ = 61 ppm. This means that the phosphate group of DCP in the L phase is not completely immobilized and that some motions average the CSA compared with the crystalline phases. However, the phosphate group is not experiencing fast rotation on the NMR time scale, as indicated by the spectrum of a nonaxial symmetry (Fig. 2 B).

The line shape of the spectrum of DCP in the L phase (Fig. 2 B) is significantly different from that of phospholipids (Fig. 2 E). When DCP is embedded into the bilayer, any intramolecular rotations of the phosphate group are restricted because it is attached to the two hydrophobic alkyl chains. In contrast to DCP, rotation of the phosphate group of phospholipids is not hindered and can contribute to the reduction of the CSA.

In liquid-crystalline bilayers, lipid molecules undergo fast rotation around the bilayer normal. For the case of fast rotation about a fixed axis, the original tensor is replaced by an effective tensor, which is axially symmetric and has the elements:

(1)

(2)

where and are the components parallel and perpendicular to the rotation axis and the angles  and  are Euler angles between the rotation axis and the principal angles of the shielding tensor (Mehring et al., 1971; Seelig, 1978).

In the case of DCP, we proposed that the direction of the element ₂₂ coincides with the bilayer normal. Therefore, fast molecular rotation will completely average the elements ₁₁ and ₃₃ and not affect the element ₂₂, resulting in equations:

(3)

(4)

(5)

The anisotropy which is observed depends now only on the values of _ii. In the L phase, the intramolecular rotations of the DCP phosphate group are restricted for the same reason as in the L phase. Therefore, the CSA of DCP in the L phase can be calculated assuming that fast molecular rotation is the main contribution in the additional averaging of the CSA compared with the L phase. For this, the values of the partially averaged shielding tensor in the L phase of DCP were used to calculate of DCP in the L phase (Eqs. 3-5). The calculated value of 15 ppm is in a good agreement with the experimental data. The simulated and experimental spectra of DCP in the L phase are shown in Fig. 7 C.

³¹P NMR spectra of DCP in the L phase exhibit a significantly smaller anisotropy compared with phospholipids, although the motional freedom is more restricted in the case of DCP. The reason for this is in the different orientation of the shielding tensor of DCP and phospholipids with respect to the lipid bilayer. Polar groups of phospholipids are aligned essentially parallel (within 30°) to the plane of the bilayer (Scherer and Seelig, 1989), whereas both hydrocarbon chains attached to the phosphate group of DCP are embedded in the bilayer perpendicular to the bilayer surface (Fig. 7, A and B).

DCP stabilizes the L phase and destabilizes the L lamellar phase in monostearoylglycerol

An interesting feature of monoglyceride/water systems is the ability to form a great variety of different phases. In contrast to phospholipids, phase transformations of monoglycerides occur in a relatively narrow temperature range. Qualitatively, the phase behavior of monoglycerides can be easily understood by relating the packing properties of lipid molecules to their shape (Israelachvili et al., 1977). The headgroup of monoglycerides consists of a glycerol moiety. Because of their low hydration capacity (Morley and Tiddy, 1993), the size of the headgroup is almost the same in different phases except in the coagel, in which water is not present. The dynamically averaged volume of the acyl chain increases upon heating because of chain rotation and trans/gauche isomerization along the chain inducing the phase transitions. When lipid molecules have a cylindrical shape, they are packed in a bilayer structure. Upon heating, the acyl chain volume increases and molecules now have more of a cone-like shape with the headgroup at the pointed end. The self-packing of such cone-like molecules can lead to the formation of highly curved cubic phases or inverted phases with a negative curvature.

The phase behavior of the MSG:DCP mixture can likewise be related to the molecular shape of these lipids. Fig. 8 illustrates the difference in the molecular shape of MSG and DCP. The molecule of DCP consists of a relatively small headgroup with two attached alkyl chains. Therefore, the dynamically averaged molecular shape of DCP will be cone-like. Being placed into a flat bilayer will increase the propensity of lipids to be organized into inverted structures with a negative curvature (de Kruijff, 1997a, b). Indeed, when DCP is present the [²H₃₅]-MSG:DCP mixture does not form a "fluid" liquid-crystalline bilayer, but directly transforms from the L into the L₂ phase instead.

View larger version (62K): [in this window] [in a new window]   FIGURE 8   Molecular models of MSG (A) and DCP (B) in the extended conformation. The dynamically averaged molecular shape is illustrated by the surrounding lines. Light spheres represent hydrogen atoms. Dark spheres represent carbon, oxygen, and phosphorus atoms.

Another effect of DCP on the phase behavior of [²H₃₅]-MSG is the stabilization of the metastable L phase. This can be understood considering the way the L_c phase is thought to form. Crystallization of the L phase into the coagel occurs via expelling of water and formation of hydrogen bonds between monoglyceride molecules in adjacent bilayers. The presence of the negatively charged DCP will inhibit the bilayers to come in a close proximity, thereby preventing crystallization of the monoglyceride bilayers.

Water-soluble proteins stabilize the L lamellar phase in [²H₃₅]-MSG:DCP mixture

The water-soluble -lactoglobulin and lysozyme change the phase behavior of the monoglyceride dispersion when DCP is present, demonstrating that DCP is responsible for lipid-protein interactions in this system. Although incorporation of DCP in [²H₃₅]-MSG destabilizes the L lamellar phase, in the presence of the proteins, this effect is completely eliminated. Because -lactoglobulin and lysozyme do not affect the phase behavior of pure [²H₃₅]-MSG, this suggests that electrostatic interactions between the proteins and the negatively charged DCP are involved. Also, it was found that a high ionic strength decreases the stabilizing effect of these proteins on the L phase of [²H₃₅]-MSG:DCP. However, the total charge of protein does not seem to be important for interaction of the protein with the negatively charged lipid-water interface, as -lactoglobulin has a negative net charge and lysozyme has a positive net charge. This implies that electrostatic interactions must take place on the level of the positively charged amino acid residues of the proteins. In contrast, in the presence of these proteins, the L phase of the [²H₃₅]-MSG:DCP mixture is observed up to higher temperatures than in pure [²H₃₅]-MSG. This can not be explained by only electrostatic interactions, because the simple neutralization of the electrical charge of DCP would instead rather destabilize the L phase. The mechanism and consequences of lipid-protein interactions in [²H₃₅]-MSG:DCP can be understood if one takes into account both electrostatic interactions and the special effect of nonbilayer-forming lipids on liquid-crystalline bilayers (de Kruijff, 1997a, b; Van den Brink-van der Laan et al., 2001). This is illustrated in Fig. 9.

View larger version (26K): [in this window] [in a new window]   FIGURE 9   Protein insertion into the MSG:DCP bilayer. Being placed into a bilayer, a nonbilayer-forming DCP creates a space at the interface (A). In the absence of proteins, the flat liquid crystalline bilayer enriched with DCP transforms into the inverted phase with negative curvature (B). When protein is inserted into the bilayer the volume of the headgroup region is increased, effectively neutralizing the tendency of DCP to be organized in inverted structures (C).

According to this model, both -lactoglobulin and lysozyme interact with the [²H₃₅]-MSG:DCP interface. Although lysozyme and -lactoglobulin differ in the sign of the net charge, they have almost the same number of positively charged residues, which can electrostatically interact with the negatively charged DCP. In addition, the relatively small headgroup of DCP creates a space at the interface, thereby facilitating protein insertion (Fig. 9 A). This is in agreement with our observation that the presence of DCP or dioctadecylphosphate facilitates insertion of -lactoglobulin and lysozyme into monoglyceride monolayers (Boots et al., 1999, 2001). In line with this model is also the observation that insertion of proteins may destroy the acyl chain packing order of [²H₃₅]-MSG at the lipid/water interface, as indicated by the line shape changes in ²H NMR spectra (Fig. 6). In the absence of proteins, the flat bilayer enriched with a nonbilayer-forming lipid DCP (Fig. 9 A) transforms into the inverted phase (Fig. 9 B). However, when protein is inserted into the bilayer the volume of the headgroup region is increased neutralizing the tendency of DCP to be organized in inverted structures (Fig. 9 C).

At low temperature the presence of -lactoglobulin and lysozyme does not accelerate transformation of the thermodynamically metastable L phase of nonsonicated [²H₃₅]-MSG:DCP dispersions into the coagel. This is in contrast to the results obtained for sonicated dispersions (Boots et al., 1999, 2001), in which we showed that -lactoglobulin and lysozyme induce fast crystallization of small sonicated [²H₃₅]-MSG:DCP L phase vesicles into the coagel at 20°C. A likely explanation for this apparent discrepancy is that the lipid molecules are less tightly packed at the interface of the outer monolayer of highly curved sonicated vesicles compared with the flat, extended bilayers of nonsonicated dispersions (Huang and Mason, 1978). This may facilitate interaction of proteins with the outer monolayer of sonicated vesicles. Indeed, -lactoglobulin and lysozyme readily interact with sonicated vesicles of [²H₃₅]-MSG:DCP at 20°C, inducing disruption of the lipid bilayer (Boots et al., 1999). The following rearrangement of the lipid molecules results in the formation of the coagel, which is thermodynamically stable under these conditions. In addition, nonsonicated dispersions, which consist of randomly oriented extended bilayers, are much more viscous. Stacking of these bilayers into the L_c phase can only proceed after mechanical disruption of the bilayers, which slows down the rate of crystallization. In line with this explanation is the observation that shearing of the monoglyceride gel accelerates the coagel formation (Cassin et al., 1998).