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J. Phys. Chem. B 1999, 103, 8908-8914
Surfactant-Induced Alterations of Lecithin Molecular Dynamics in Bilayers Studied by Quasielastic Neutron Scattering and Solid-State NMR Christine Gliss,† Helene Casalta,‡ and Thomas M. Bayerl*,† Institut fu¨ r Experimentelle Physik V, UniVersita¨ t Wu¨ rzburg, 97074 Wu¨ rzburg, Germany, and Institut Max Von Laue-Paul LangeVin, AVenue des Martyrs, 38042 Grenoble Cedex 9, France ReceiVed: May 6, 1999; In Final Form: August 27, 1999
Quasielastic neutron scattering (QENS) measurements were employed to study changes in high-frequency dynamics of dipalmitoyl-phosphatidyl-choline (DPPC) bilayers induced by small amounts of nonionic surfactants (tetra-ethyleneglycol-mono-n-dodecyl ether, C12E4). The experiments were performed at three energy resolutions probing different frequency domains (GHz to lower THz range) of molecular motion and at two temperatures, corresponding to the crystal-like gel phase (T ) 20 °C) and the fluid phase (T ) 50 °C) of the bilayer. Two orientations of the bilayer stack were studied to obtain information about the anisotropy of the dynamics with respect to the in-plane and the out-of-plane lipid motion. At 5 mol % surfactant in a fluid DPPC bilayer, we observed drastic changes of lipid dynamics in the frequency domain which is dominated by diffusive motions of the whole molecule. The presence of surfactant increased the lipid in-plane diffusion constant by 50% and the spatial extension of this motion by 25%. In contrast, the out-of-plane lipid motion showed a reduction of the diffusion constant by 60% and its spatial extension was reduced by 40%. Solidstate deuterium NMR of fluid DPPC bilayers showed that the surfactant caused a reduction of the order parameter of the lipid chains and changed the shape of the order parameter profile. In the high-frequency domain where kink motions of the lipid chains dominate the dynamics, no surfactant effects were observed. In a time averaged picture, the results suggest a surfactant-induced spread of the lipid chains in the bilayer plane and a concomitant reduction of bilayer thickness. For gel phase bilayers, no surfactant- induced alterations of lipid dynamics were detected.
Introduction The ability of nonionic surfactants to solubilize lipids and other biomolecules in mixed micelles is well established1 and has important applications in biochemistry for protein reconstitutions in vesicles.2 Although knowledge of the solubilization process at the molecular level is still incomplete, there is ample information available about surfactant-lipid interactions at the high surfactant proportions under which solubilization occurs.3 Another feature of surfactant-lipid interaction is even less well studied. Nonionic surfactants at subsolubilizing concentrations can intercalate spontaneously into lipid bilayers4-6 and biological membranes7 without destroying the host matrix. However, they can drastically change physical properties of the membrane such as the bending modulus,8 phase transition behavior,9 morphology10 and the activity of membrane proteins.3 It is noteworthy that significant changes are observed even at low proportions of surfactant.11 Although thermodynamic12,13 and structural14 features of this process have been studied recently in great detail, information about the dynamical consequences of surfactant-membrane interaction is virtually not available yet. However, this information is crucial for understanding the surfactant effects from a molecular dynamic point of view. * Address correspondence to: Prof. Thomas M. Bayerl, Universita¨t Wu¨rzburg, Physikalisches Institut EP-5, D-97074 Wu¨rzburg, Germany. Phone: ++49-931-888 5863. Fax: ++49-931-888 5851. E-mail: bayerl@ physik.uni-wuerzburg.de. † Universita ¨ t Wu¨rzburg. ‡ Institut Max von Laue-Paul Langevin.
Pure lipid dynamics in fluid and gel phase bilayers has been studied up to the terahertz frequency range by quasi-elastic neutron scattering (QENS).15-17 The uniqueness of QENS for such studies lies mainly in providing the full autocorrelation function of the molecular motion (i.e., both spatial and temporal correlation), in allowing the experimenter to adjust different experimental time scales and length scales and in the option to mask unwanted dynamical modes by selective deuteration of the molecules under study. Furthermore, by using oriented lipid membranes QENS can provide unique information about the anisotropy of molecular motions. Finally, the time scale of QENS matches with that of state-of-the-art molecular dynamics (MD) simulations of membranes and thus allows a direct comparison between experimental and theoretical results. From the vast variety of chemically pure surfactants available, we have chosen a nonionic surfactant of the polyethylene-glycolmonododecyl-ether variety (C12En) with four oxyethylene groups (n ) 4) and have studied the dynamical consequences of its intercalation at low proportions in fully hydrated oriented multilayers of a synthetic lecithin (dipalmitoyl-phosphatidylcholine, DPPC) at two temperatures (20 and 50 °C) corresponding to the gel and fluid phase of the bilayer. The effect of C12E4 on the structure and thermodynamics of lecithin bilayers and monolayers has been studied previously by other authors.10,14 Our specific interest was to search for those alterations in the high-frequency dynamics of the DPPC molecules due to the presence of surfactant which may provide an explanation of the above-mentioned changes of membrane properties, in particular the softening of membranes. Moreover, we wanted to restrict our study to the case of low surfactant concentrations
10.1021/jp991505i CCC: $18.00 © 1999 American Chemical Society Published on Web 10/06/1999
Dynamics of Lecithin/Surfactant Bilayers
J. Phys. Chem. B, Vol. 103, No. 42, 1999 8909
in the membrane where other, more structure-oriented, physical methods cannot detect significant changes of the physicochemical membrane properties. Materials and Methods Materials and Sample Preparation. 1,2-dipalmitoyl-d31glycero-3-phosphocholine-d13 (DPPC-d75) was purchased from Avanti Polar Lipids (Alabaster, AL). The surfactant tetraethyleneglycol-mono-4-dodecyl ether (C12E4) was obtained from Nikko Chemical Co. Ltd. (Tokyo, Japan). Highly purified D2O was purchased from Deuchem (Leipzig, Germany). The solid substrates for multilayer preparation were highly polished, undoped silicon wafers of 150 µm thickness and 5.0 cm diameter which were purchased from Virginia Semiconductors (Fredericksburg, VA). The oriented multilayer samples of DPPC containing 5 mol % C12E4 and of pure DPPC were prepared according to procedures described in detail previously.15 Each sample consisted of a stack of 9-10 wafers with about 3000 bilayers sandwiched between adjacent wafers, giving a total of 0.4 g lipid per sample. The hydration of the multilayers (20 wt % water) was achieved by equilibrating them against a saturated heavy water (D2O) vapor at elevated temperature (T ) 60 °C) for a minimum of 30 h before stacking them. After this the sample was annealed in saturated vapor at T ) 80 °C for 10 h. Then the samples were transferred to a gastight aluminum sample holder for the QENS measurements. For deuterium NMR measurements the samples were prepared analogously with the exception that the hydration was done using deuterium-depleted water and that the silicon substrates were cut in rectangular shapes to fit into a gastight 10 mm diameter NMR sample tube (cf. ref 18 for details). Methods. QENS. Since QENS is a method rarely used, a few basic features of dynamic neutron scattering should be recalled here. In neutron scattering, two scattering cross-sections have to be distinguished, the coherent and the incoherent crosssection. The former probes the pair correlation function between different scatterers in a sample and thus provides both structural information and correlated dynamics. Incoherent scattering probes the autocorrelation function of the scattering particles, i.e., the molecular dynamics of the molecules. The possibility of distinguishing the two cross-sections makes neutron scattering a unique tool for the determination of dynamical parameters. A deliberate distinction between the two types of scattering is possible by simply choosing a “convenient” scatterer in the sample. For biological applications the most important incoherent scatterer is the hydrogen atom. The deuteron scatters mainly coherently but much more weakly than the incoherent scattering of the proton. Thus (selective) deuteration offers the means to mask (parts of) molecules in incoherent scattering experiments. In our case, the surfactant concentration was chosen in such a way that masking the surfactant was not necessary because of the relatively small number of protons in the surfactant compared to the DPPC. For hydration of the membrane stack, deuterated water (D2O) was used. Therefore, we can assume our scattering signal to be incoherent scattering arising from DPPC. The time scale on which the correlation functions are probed is determined by the energy resolution of the spectrometer used. Hence, by employing various energy resolutions, different time domains of the correlation function can be probed selectively. We used the IN10 and IN16 backscattering spectrometers and the IN5 time-of-flight spectrometer at the Institute Max von Laue-Paul Langevin (Grenoble, France). For both backscattering spectrometers, energy analysis was done by unpolished Si (111) crystals at an energy resolution of 1 µeV and the incident
Figure 1. Schematic depiction of the scattering geometry used for QENS of oriented membrane stacks for the case β ) 135°. The momentum transfer Q is shown with its components parallel and perpendicular to the membrane normal n; ki and kf are the incoming and the final neutron beam. The angles are connected in quasielastic approximation by cos(R) ) cos(90° - β - θ).
TABLE 1: Summary of the Experimental Conditions under which QENS Measurements on DPPC/ C12E4 Oriented Multilayers Were Performed (see text for details) instrument (resolution) T (°C) 20 50
IN10/IN16 (Γ ) 1 µeV)
IN5 (Γ ) 19 µeV)
IN5 (Γ ) 63 µeV)
135° orientation 45° orientation 135° orientation 45° orientation
135° orientation
135° orientation
135° orientation
135° orientation
wavelength was 6.271 Å. The time-of-flight spectrometer was used at an incident wavelength of 9 and 6 Å, giving an energy resolution of 19 and 63 µeV, respectively. To measure the anisotropy of the lipid motion and take full advantage of the use of oriented multilayer samples, two orientations of the sample in the neutron beam were studied. At an orientation of 135° between the incident neutron beam and the membrane normal () normal to the silica wafer), the momentum transfer is directed predominantly perpendicular to the membrane normal (i.e., parallel to the bilayer plane). In this case, the in-plane motion of the lipid will dominate the incoherent scattering. In contrast, at an orientation of 45°, the momentum transfer is directed predominantly parallel to the membrane normal and thus the incoherent scattering is dominated by out-of-plane motion of DPPC along the membrane normal. The scattering geometry is schematically depicted in Figure 1, and the experimental conditions are summarized in Table 1. The in-plane motion (135° orientation) as well as the out of plane motion (45° orientation) was measured by IN10 (energy resolution 1 µeV) above and below the phase transition (T ) 20 °C and T ) 50 °C). For the higher frequency motions detected by IN5 (energy resolutions Γ ) 19 µeV and Γ ) 63 µeV), we only measured the in-plane motion at the two temperatures. Standard ILL procedures for background and cell correction as well as normalization of the incoherent scattering to a vanadium standard sample were applied. 2H NMR. Deuterium NMR experiments were performed using a Bruker AMX-500 spectrometer equipped with a 10 mm solidstate probe at 50 °C. The sample was oriented with its normal parallel to the magnetic field. The spectra were obtained by applying a quadrupolar echo (QE) pulse sequence with two 90° pulses of 5.5 µs duration, a pulse separation of 35 µs, and a CYCLOPS phase cycling sequence. The repetition time was
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TABLE 2: Model-Independent Long-Range Diffusion Coefficients Dlat of DPPC with and without 5 mol % C12E4 in the Plane of the Bilayer (135° Orientation) Obtained under Fluid (T ) 50 °C) and Gel (T ) 20 °C) Phase Conditions from the IN10 Data Fits Shown in Figure 2
gel phase (T ) 20 °C) fluid phase (T ) 50 °C)
sample
long range diffusion Dlat (10-11 m2/s)
DPPC DPPC + C12E4 DPPC DPPC + C12E4
7(1 7(2 22 ( 3 35 ( 4
TABLE 3: Results of the Model-Dependent Data Analysisa Dlat Dnor h Dcirc r (10-11 m2/s) (10-11 m2/s) (Å) (10-11 m2/s) (Å) DPPC T ) 20 °C) DPPC + C12E4 (T ) 20 °C) DPPC (T ) 50 °C) DPPC + C12E4 (T ) 50 °C)
0.8 ( 0.1
2 ( 0.4
1.4
22 ( 4
1.5
0.8 ( 0.2
2.6 ( 0.5
1.4
13 ( 3
1.4
1.9 ( 0.4
36 ( 5
4
200 ( 30
4.2
3.3 ( 0.5
10.5 ( 3
2.5
300 ( 35
5.4
a For the DPPC in-plane motion (135° orientation), a superposition of long-range diffusion (giving Dlat) and restricted diffusion in a circle of radius r (giving Dcirc) was applied. For DPPC motion along the membrane normal (45° orientation), a diffusion between two walls separated by a distance h (giving Dnor) was considered. The errors for the r and h values given in the table are 10%.
150 ms, and 4096 complex data points were collected in quadrature with a dwell time of 1 µs. Data Analysis. Two types of procedures were applied to fit the QENS data. These have been described in detail previously15 and characterized as the “single spectrum fitting procedure” and the “simultaneous fitting procedure”. The dynamic structure factor S(Q, ω) of each motion considered is represented by a sum of Lorentzians (plus an elastic line for motions restricted in space). For a superposition of independent motions contributing to the scattering, the resulting S(Q, ω) is the convolution of the corresponding dynamic structure factors for each motion. Fitting the IN10 data to a single Lorentzian () one quasielastic line) gave a model independent measure of the translational “long-range” diffusion coefficient D as listed in Table 2. Note that the term “long-range” refers to any translations over a length of more than 8 Å, the experimental length scale determined by the Q-range covered. The second step in data evaluation was to find a model for the motion that is compatible with the observed Q-dependencies of the elastic incoherent structure factor (EISF) and line width. The model function convoluted with the instrument resolution function is then fitted simultaneously to all spectra. The free parameters are the model parameters as listed in Table 3. For IN10 data, local and longrange diffusion were used simultaneously to describe the data. For the analysis of the IN5 data obtained at high energy resolution (19 µeV), the model of a spatially restricted diffusion was used. The quasielastic broadening caused by such local diffusion was then used as a constant and separate contribution in the IN10 data analysis. For the low resolution IN5 data analysis (63 µeV), the model of kink diffusion was used.16 The Debye-Waller factor describes motions, which are too fast to be resolved in the time domain used. It was considered in the data analysis analogously to the case described in ref 15. Results The dynamics of DPPC in the presence and absence of low nonionic surfactant concentrations was measured at three
partially overlapping frequency regimes determined by the energy resolution of the QENS setup. Correspondingly, fast lipid dynamics was studied at a low resolution of Γ ) 63 µeV using the time-of-flight IN5 spectrometer while motions having longer correlation times were detected by a combination of IN5 (Γ ) 19 µeV) and the backscattering spectrometers IN10/IN16 (Γ ) 1 µeV). These various energy resolutions have been demonstrated previously to allow a comprehensive description of lipid motions covering the range from lateral lipid diffusion at the low-frequency side (GHz range) to kink motion of the chains at high frequencies (lower THz range).15,16 Furthermore, all IN10/IN16 measurements were done at two orientations of the of the sample normal () normal direction of the oriented lipid multilayers) with respect to the incident neutron beam: Experiments performed at 135° orientation essentially detected lipid motions in the plane of the bilayer, while those done at 45° had the momentum transfer directed along the membrane normal and thus were sensitive to the out-of-plane lipid motion.15 IN5 measurements were performed for the 135° orientation only, since previous studies of DPPC did not indicate motional anisotropies at these high frequencies. The experimental conditions are summarized in Table 1. Slow Lipid Motion (1 µeV Resolution, IN10/IN16 Results). The effect of the surfactant was clearly observed for lipid motions at lower frequencies (energy resolutions of 1 µeV (IN10, IN16) and of 19 µeV (IN5)), predominantly for the fluid bilayer phase. Additionally, we observed an orientation dependence of the surfactant effect on the DPPC dynamics measured in this frequency range (0.2-300 GHz). Lipid In-Plane Motion (135° Orientation). Figure 2A compares the width of the quasielastic line (IN16 data at Γ ) 1 µeV) obtained for pure DPPC and DPPC/ C12E4 (5 mol %) under fluid bilayer conditions (T ) 50 °C). It is obvious that the presence of the surfactant causes broadening of the QENS spectra which increases with increasing Q. Over a wide Q-range, the quasielastic line width is linear in Q2, indicating a longrange diffusive motion. A linear fit (dashed lines) gives longrange diffusion coefficients Dlat according to the Γ ) DlatQ2 dependence as listed in Table 2. This model-independent approach shows that the presence of the surfactant drastically increases the value of Dlat for DPPC from 22 to 35 10-11 m2/s. Deviations of the curve from linear dependence at high Q values may indicate the presence of another lipid motion which is, in contrast to the lateral diffusion, spatially confined within a limit below the length scale of the experiment (8 Å). Deviations from the linear behavior at the low Q end are due to the fact that at these Q values the diffusion-induced line broadening is below the energy resolution (1 µeV) of the spectrometer. To bypass this instrumental resolution problem, the theoretical point Γ ) 0 at Q ) 0 was included in the fits for the determination of Dlat. In contrast to the fluid phase results, no surfactant effects on the diffusive motion can be observed for the gel phase bilayer (Figure 2B). Long-range diffusion coefficients determined from the linear fits (again assuming Γ ) 0 for Q ) 0) are at least a factor of 3 lower. Analyzing the EISF vs Q dependence gives information about the spatial restriction of the diffusive lipid motion dominating the frequency range covered by IN10/IN16. In Figure 3A the EISF vs Q dependence for the in-plane measurement (135° orientation) at the two temperatures is compared with those of motional model calculations. The data show clearly the much faster long-range DPPC diffusion for the fluid phase (T ) 50 °C) by the significantly steeper decrease of the EISF with
Dynamics of Lecithin/Surfactant Bilayers
Figure 2. A. Plot of the line width of the quasielastic line obtained at IN16 (energy resolution Γ ) 1 µeV) at T ) 50 °C and 135° orientation for pure DPPC (open circles) and for DPPC/ C12E4 (5 mol %, full circles). Note that the point at origin is based on a theoretical assumption. The dashed lines represent linear fits the slope of which gave the long-range lateral diffusion coefficients of Dlat listed in Table 2. B. same as in A but for a gel phase bilayer (T ) 20 °C). Open circles represent pure DPPC data and full cicles DPPC/ C12E4 data.
increasing Q values. Moreover, the additional increase in Dlat by the presence of the surfactant results in an even more pronounced EISF decrease. In contrast, for the gel phase (T ) 20 °C) the EISF decreases much more slowly with Q, indicating the dominance of spatially restricted diffusion and there is no surfactant effect. For the model analysis, we have employed the same models which were established previously for the pure DPPC dynamics studied by IN10.15,16 These models considered the following lipid motions spatially restricted in the bilayer plane: (a) diffusion in a circle of radius r with a diffusion coefficient Dcirc, (b) two-site jumps, and (c) diffusion between two walls of distance l with a local diffusion coefficient Dloc. Long-range lateral diffusion was superimposed on all restricted motions. We found that model (a) described the data best in both fluid and gel phase. Figure 3A shows model fits to the data for the “diffusion in a circle” model (characterized by Dcirc and the circle radius (r) superimposed on long-range lateral diffusion Dlat. The value of Dlat was taken from the model independent line width analysis (Table 2). This combined model gave the best fits to the data in both fluid and gel phase. The values of Dlat, Dcirc, and r extracted from this model are listed in Table 3. For the in-plane diffusion, this model-dependent data analysis indicates a significant increase in the spatial extension r (by 25%) and in Dcirc (by 50%) caused by the presence of the surfactant in the fluid phase bilayer. For the gel phase bilayer,
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Figure 3. EISF vs momentum transfer Q of DPPC oriented multilayers with (full symbols) and without (open symbols) 5 mol % C12E4 measured by IN10 (Γ ) 1 µeV) under fluid (T ) 50 °C, circles) and gel phase (T ) 20 °C, squares) conditions. The dashed lines represent fits to the data in terms of a motional model (see text for details), giving diffusion coefficients and spatial restrictions as listed in Table 3. A. 135° orientation (in-plane motion); B. 45° orientation (out-of-plane motion in normal direction).
the model suggests a slight reduction in Dcirc at an unchanged spatial extension r and long-range diffusion Dlat. Lipid Out-of-Plane Motion (45° Orientation). In contrast to the in-plane measurements, a plot of the quasielastic line width Γ vs Q2 for the out-of-plane motion did not give any linear dependence at both temperatures (data not shown). This was not unexpected since the lipid motion along the membrane normal is spatially restricted by its confinement within the monolayer leaflet. Under gel phase conditions (T ) 20 °C), the presence of the surfactant does not change the Γ vs Q dependence. For the fluid bilayer (T ) 50 °C) the surfactant causes a significant decrease of Γ for Q > 1 Å-1. Analysis of the EISF provides some deeper insight into the restricted DPPC motion in normal direction. Figure 4B shows the Q-dependence of the EISF at 45° orientation and for the two temperatures. Even without any motional model it is obvious from the data that the presence of the surfactant in the fluid bilayer (T ) 50 °C) increases the EISF and thus the spatial restriction of the DPPC motion. Note that this represents the opposite behavior to what was observed for the in-plane motion. In contrast, for the gel phase bilayer the EISF shows no dependence on the presence of surfactant. The motional models considered for the out-of-plane motion were restricted diffusion in the normal direction between two
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Figure 5. Deuterium NMR order parameter profile of chain perdeuterated DPPC-d62 in oriented multilayers (bilayer normal parallel to the magnetic field) without (open circles) and with (full circles) 5 mol % of C12E4 at T ) 50 °C. Figure 4. EISF vs momentum transfer Q (135° orientation) of DPPC without (open symbols) and with (full symbols) 5 mol % C12E4 measured by IN5 at an energy resolution of Γ ) 63 µeV for a fluid phase (T ) 50 °C, circles) and a gel phase (T ) 20 °C, squares) oriented multilayer.
walls of separation length h, giving a diffusion coefficient Dnor, and two-site jumps in normal direction of jump length l and jump rate κ. Both models gave acceptable fits to the data of Figure 3, but the diffusion between walls model proved slightly superior for the fluid phase data. The results of the model calculations are listed in Table 3. These results suggest that, at T ) 50 °C, the presence of the surfactant causes a reduction both in Dnor (down to 30% of the value for pure DPPC) and in h (by a factor of 2 compared to pure DPPC). This slow down of diffusion is in agreement with the observed reduction of quasielastic line broadening for the surfactant containing sample at 50 °C. Similarly, the increased spatial restriction of out-ofplane motion in the presence of surfactant was obvious from the significant increase of the EISF of the fluid mixture (Figure 3B). Fast Lipid Motion (63 µev Resolution, IN5 Results). For high-frequency dynamics measured by IN5 at Γ ) 63 µeV, we obtained a rather unexpected result: The plot of the EISF vs Q for DPPC oriented multilayers with and without C12E4 (Figure 4) shows that the geometry of the motion in the plane of the bilayer (135° orientation) was virtually unaffected by the surfactant at both temperatures. Since the frequency range covered by these measurements (up to 0.3 THz) is dominated by kink motion for pure DPPC, we can conclude that the surfactant does not alter the spatial extension of this motion, and thus the average number of kinks per lipid chain. This conclusion is further supported by the finding that the line width of the quasielastic line with and without surfactant was identical within the error of the measurements. Hence, the presence of the surfactant did not alter the correlation times of the kink motions dominating this high-frequency range. Since the surfactant caused no measurable effect on the highfrequency motion of DPPC, we have refrained from presenting model fits to the data. Motional models for IN5 data of pure DPPC at both temperatures have been discussed in detail previously.16 Our results obtained with the pure DPPC reference sample were in perfect agreement with those published previously. Orientational Order of the Lipid Chains: Deuterium NMR. 2H NMR measurements of oriented multilayer samples of chain perdeuterated DPPC-d62 were performed with and without C12E4 (5 mol %) under fluid phase conditions (T ) 50 °C). Similar 2H NMR data have been reported for mixtures of
a related nonionic detergent, C12E8, with phospholipids.5,6 The order parameter profiles extracted from the oriented spectra (bilayer normal parallel to the magnetic field) are shown for both cases in Figure 5. The presence of the surfactant caused a significant decrease of molecular order for all chain methylene positions except the one next to the glycerol backbone. This effect became even more pronounced toward the end of the chain. Most interestingly, the characteristic DPPC order parameter plateau which extends from the C-1 to the C-9 position for fluid phase DPPC19,20 was completely abolished by the surfactant. Instead, the C12E4 caused the DPPC order parameter to decrease linearly with the chain position, as also observed upon icorporation of C12E8 into phospholipid bilayers.21 To appreciate this drastic effect one has to keep in mind that the 2H NMR time scale is in the lower µs range and thus more than 4 orders of magnitude slower than the IN10 time scale. Discussion The most striking dynamical consequences of the presence of C12E4 in the DPPC bilayers are as follows: (1) While fluid phase DPPC exhibits drastic changes of its dynamic properties, no significant effects were observed for the gel phase. (2) For fluid bilayers, both long-range and restricted lipid diffusion were affected. In the bilayer plane, the surfactant increased Dlat and Dcirc in a similar proportion as well as the spatial confinement r of the restricted diffusion. In contrast, the restricted diffusion Dnor in the normal direction and its spatial extension h was decreased by the surfactant. (3) No surfactant effects were observed in the high-frequency regime where kink motions dominate the DPPC dynamics. (4) At the comparatively very low frequencies of the 2H NMR time scale (10-6 s), a drastic reduction of the molecular order of the DPPC palmitoyl chains was observed. In this first QENS study of DPPC/C12E4 mixtures we wish to concentrate on how low concentrations of a typical nonionic surfactant can alter the DPPC dynamics. To keep this discussion focused, we will refrain from a detailed discussion of pure DPPC bilayer dynamics which has been discussed for this frequency range in great detail elsewhere.15 The model-dependent data analysis in terms of a restricted DPPC diffusion coefficient Dnor in a cylindrical volume of height h (oriented along the membrane normal) and of radius r (Table 3) was already successfully applied in a previous study for the assessment of the hydration effects on lipid dynamics.16 Our present results indicate that this model can also adequately describe the modulation of DPPC dynamics by low concentra-
Dynamics of Lecithin/Surfactant Bilayers tions of C12E4. In terms of this model, the surfactant effect on fluid phase DPPC is essentially a reshaping of the cylinder occupied by the lipid while preserving its volume, superimposed by a higher long-range lateral diffusivity of the whole molecule (i.e. of the cylinder). The reshaping consists of a shrinkage of the cylinder height (directed parallel to the membrane normal) and a corresponding extension of its radius, i.e., in a timeaveraged picture the molecular area of DPPC projected to the membrane plane increases while the bilayer thickness decreases. The observed increase of molecular area in the bilayer plane that is occupied by the diffusing DPPC is in good agreement with X-ray diffraction results,14 where an area increase of 10% was observed at 5 mol % C12E4. Furthermore, film balance studies of DPPC monolayers with C12E4 gave similar increases in the DPPC molecular area in the fluid (LE) phase.22 Moreover, a concomitant reduction of bilayer and monolayer thickness by C12E4 was observed by X-ray diffraction for palmitoyl-oleoylphosphatidylcholine (POPC)14 and by neutron reflection for DPPC monolayers.23 The thinning of the bilayer by the surfactant can also explain a reduction of the 2H NMR order parameter. It has been demonstrated that the molecular order parameter of a lipid chain scales with the hydrophobic thickness of the bilayer.24 However, the thinning alone does not provide a conclusive explanation for the complete abolition of the “plateau region” of the order parameter profile by C12E4 (cf. Figure 5). It was shown previously that the QENS data of pure DPPC long-range diffusion15 strongly support the free-volume model of lateral diffusion.25 In terms of this model, the observed increase of Dlat in the presence of C12E4 at 50 °C can be understood as a consequence of the creation of additional free volume by the surfactant. The substantial area per molecule occupied by C12E4 in fluid phase DPPC of 94 Å2 22 (DPPC is about 80 Å2 under these conditions) may explain the creation of additional free volume. To appreciate this, it should be noted that the surfactant area is dominated by the hydration of its bulky (poly)-ethylene-oxide headgroup while its tail cross-section is comparatively small, that the whole molecule is at least 30% shorter than DPPC, and that it protrudes deeper into the water phase.23 Hence, significant free volume is created in the hydrophobic tail section of those DPPC molecules adjacent to C12E4 molecules, thereby allowing the fluid lipid chains to diffuse into this additional volume. Since the average distance between adjacent molecules in the bilayer and the experimental length scale above which all diffusive motion is seen as longrange are similar (7-8 Å), such a diffusive motion would contribute to both Dlat and Dcir. Indeed, a significant increase of both diffusion coefficients is suggested by the modeldependent data analysis (Table 3). Moreover, the widening of the area accessible to the DPPC chains should reduce the attractive van der Waals interactions in the chain region and give rise to the loss of DPPC chain order even for those chain segments close to the glycerol backbone and thus abolish the characteristic order parameter plateau observed in pure DPPC bilayers. It is interesting to note that despite the free volume increase caused by the surfactant as indicated by the IN10 results, the IN5 measurements suggest that the number of gauche kinks per lipid remains rather constant. To reconcile these contradictory results, one has to appreciate that the formation of gauche kinks is a thermally driven process and largely independent of the accessible free volume as long as it is above a certain critical volume.16 However, the thermal energy per DPPC is not changed by the surfactant, and thus the average number of kinks
J. Phys. Chem. B, Vol. 103, No. 42, 1999 8913 per chain is a constant. To achieve the spreading of the chains into the adjacent voids as suggested by IN10 results, the position of kinks along the chain must change rather than their number. While for pure DPPC the probability of kink formation was found to be rather constant along the chain, the presence of the surfactant could change this, e.g., by increasing the probability of single gauche conformations close to the glycerol backbone. The latter would drastically increase the restricted diffusion constant and the spatial confinement (expressed by Dcir and r in our model, cf. Table 3) of all methylene segments below the single gauche position, without requiring an increase of the total number of kinks. Moreover, the occurrence of gauche conformers near the glycerol backbone would explain the change from a rather constant molecular order within the “plateau region” (positions C-1 to C-9 of the palmitoyl chain for pure DPPC) to a rather linearly decreasing NMR order profile (Figure 5) in the presence of the surfactant. The finding that QENS does not detect any significant effects of the surfactant on gel phase DPPC bilayer supports the above interpretation. In this phase the lipid chain order is dominated by the all-trans conformation which drastically restricts the chain segments’ translational degrees of motional freedom and thus effectively prevents their diffusion into hydrophobic voids created by the tail region of adjacent surfactant molecules. Increased DPPC chain spread in the fluid DPPC bilayer plane caused by the neighborhood of surfactant molecules may also explain the observed concomitant reduction of the DPPC outof-plane motion (Table 3). A DPPC molecule with its chains spread over a larger cross-sectional area than occupied by its headgroup will be hindered in its out-of-plane motion as a whole molecule by its entanglement with chains of adjacent molecules. As a result, both Dnor and its spatial restriction h must decrease. This molecular smoothing of the lipid out-of-plane motion by increased chain entanglement in the bilayer plane may also contribute to collective membrane motions by a tighter coupling of this molecular motion with collective modes such as the inplane or the transverse shear modes of the bilayer. Indeed, significantly higher amplitudes of collective membrane modes8 and a drastic reduction of the membrane bending stiffness26 caused by the presence of the surfactant were demonstrated experimentally by time-resolved video microscopy. Conclusions We have shown that low concentrations of nonionic surfactants in a fluid DPPC bilayer have a drastic effect on the molecular DPPC dynamics in the GHz to lower THz frequency regime. The most pronounced effects are the lateral (in-plane) extension of the area occupied by the diffusing lipid chains and the concomitant reduction of its the out-of-plane motion. The average number of kinks per lipid does not change in this process. These surfactant-induced dynamical changes of the DPPC might explain some aspects of the general softening of fluid bilayers by nonionic surfactants from a molecular dynamical point of view. However, some caution is required in connecting molecular dynamic results with macroscopic properties such as, e.g., the experimentally established drastic change of the membrane viscoelastic properties by low amounts of surfactant. One should bear in mind that the time scale gap between the methods which detect bilayer elastic properties and QENS is more than 8 orders of magnitude. Acknowledgment. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the BMBF.
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