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Langmuir 1998, 14, 982-984
Asymmetric Molecular Friction in Supported Phospholipid Bilayers Revealed by NMR Measurements of Lipid Diffusion M. Hetzer, S. Heinz, S. Grage, and T. M. Bayerl* Universita¨ t Wu¨ rzburg, Physikalisches Institut EP-5, D-97074 Wu¨ rzburg, Germany Received November 24, 1997. In Final Form: January 20, 1998 The bilayer-substrate coupling in fluid bilayers of dipalmitoyl phosphatidylcholine (DPPC) on a solid support of spherical silica beads was examined by measuring the lateral diffusion of the lipids in both monolayers using a deuterium NMR relaxation technique. The results obtained at 55 °C show that the lipid diffusion constant in the monolayer facing the silica surface, D ) 7.5 × 10-12 m2/s, is slower by a factor of 2 than that in the monolayer exposed to the bulk water (D ) 14 × 10-12 m2/s). This indicates that the monolayer-monolayer coupling in fluid bilayers must be rather weak compared to the monolayer-substrate coupling across an ultrathin water film between the bilayer and the silica surface.
1. Introduction Bilayers on a solid support have been established over the past few years as useful model systems of membranes with interesting applications in bioseparation, biosensors, and surface functionalization.1, 2 Detailed knowledge of the structure and dynamics of such systems is of paramount importance for the design of new applications and for understanding the interaction of supported bilayers with particles and molecules in the bulk. Although it has been shown that supported bilayers have a number of physical properties that are similar to those of free bilayers,3-5 significant differences exist: Collective surface modes are largely suppressed by the presence of the solid support 6 with consequences for the swelling behavior of the lipids.7 For the same reason, lateral tension can be much higher in supported bilayers and can lower its molecular order and prevent the formation of certain lipid phase states found in free bilayers.8 Moreover, most supports used so far (silica, silicon, mica) are charged, giving rise to a Coulomb interaction potential between the solid surface and charged lipid moieties in the bilayer which does not exist for free bilayers.9 Finally, a supported bilayer is intrinsically asymmetric, i.e., there is only one side of the bilayer accessible to the bulk while the other bilayer leaflet faces the solid surface across an ultrathin water layer of less than 30 Å thickness. NMR studies have shown that the water in such a narrow gap between two hydrophilic surfaces is partly structured and differs significantly in its dynamics from that of free water.10 Questions as to whether these different water properties may give rise to an asymmetry of the lipid diffusivity * To whom correspondence may be addressed: phone, 49-931888-5863; fax, 49-931-888-5851; e-mail,
[email protected]. (1) Sackmann, E. Science 1996, 271, 43. (2) Loidl-Stahlhofen, A.; Kaufmann, S.; Braunschweig, T.; Bayerl, T. M. Nat. Biotechnol. 1996, 14, 999. (3) Naumann, C.; Brumm, T.; Bayerl, T. M. Biophys. J. 1992, 63, 1314. (4) Bayerl, T. M.; Bloom, M. Biophys. J. 1990, 58, 357. (5) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (6) Dolainsky, C.; Mo¨ps, A.; Bayerl, T. M. J. Chem. Phys. 1993, 98, 1712. (7) Podgornik, R.; Parsegian, V. A. Biophys. J. 1997, 72, 942. (8) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 59, 289. (9) Reinl, H. M.; Bayerl, T. M. Biochem. 1994, 33, 14091. (10) Ko¨nig, S.; Sackmann, E.; Richter, D.; Zorn, R.; Carlile, C.; Bayerl, T. M. J. Chem. Phys. 1994, 100, 3307.
between the two bilayer leaflets or whether a dominating frictional coupling between the two monolayers can cancel out such asymmetries have been thoroughly addressed by fluorescence methods (FRAP) for glass as solid support in the past.11 Interestingly, this study did not reveal any asymmetries in lipid diffusion. Here we report results obtained with an NMR relaxation method which allows measurement of the lateral diffusion in the two monolayer leaflets of a supported bilayer separately without relying on bulky molecular labels. We find that for the case of a single fluid bilayer on the spherical support of a silica bead, there is a remarkable difference in the lateral diffusion of the lipids with the slower diffusion taking place in the leaflet facing the solid surface. 2. Experimental Section Selectively deuterated DPPC-d8 was obtained as a special synthesis from Avanti Polar Lipids (Alabaster, AL). Monodisperse silica beads are a special synthesis from Degussa AG, Department of Anorganic Chemistry (Hanau, Germany). Single DPPC-d8 bilayers on a solid support of silica beads were prepared by the vesicle fusion technique described in detail elsewhere.3,4 For NMR measurements, the sample was dispersed in deuterium depleted water at a lipid concentration of 12 mg/ mL. Deuterium NMR experiments were performed using a Bruker AMX-500 spectrometer equipped with a 10 mm solid-state probe at a temperature of 55 °C. The quadrupolar echo (QE) and the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences together with an appropriate phase cycling scheme12 were used for these experiments. The CPMG echo intensities were determined from the oriented spectra which were calculated by a numerical dePakeing algorithm.13 All other parameters of the measurement and methods of data analysis were described in detail previously.14
3. Results 2
Figure 1 shows H-NMR spectra of selectively chain deuterated DPPC-d8 at 55 °C (fluid phase) in two different morphologies: free multilamellar vesicles (MLV) and single bilayer on a silica bead (solid supported vesicles, SSV). Beside the line shape distortion of the MLV (11) Merkel, R.; Sackmann, E.; Evans, E. J. Phys. (Paris) 1989, 50, 1535. (12) Rance, M.; Byrd, A. J. Magn. Reson. 1983, 52, 221. (13) Sternin, E.; Bloom, M.; MacKay, A. L. J. Magn. Reson. 1983, 55, 274. (14) Ko¨chy, T.; Bayerl, T. M. Phys. Rev. E 1993, 47, 2109.
S0743-7463(97)01281-X CCC: $15.00 © 1998 American Chemical Society Published on Web 02/13/1998
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Langmuir, Vol. 14, No. 5, 1998 983
Figure 1. 2H-NMR spectra of DPPC-d8 at 55 °C as multilamellar vesicles (bottom spectrum) and single bilayers on a spherical solid support of silica (top spectrum).
Figure 3. (a) Semilogarithmic plot of the CPMG signal intensity of signal B versus time for different values of τ as indicated for t e 500 µs. (b) Plot of 1/T2CPMG(τ) versus τ2 for signals A (squares) and B (circles) for the τ values given in Figure 3a.
Figure 2. (a) Series of CPMG spectra of single DPPC-d8 bilayers on a spherical silica support for different times t (40, 100, 160, 220, 300 µs from top) at τ ) 20 µs. (b) De-Paked spectra from Figure 2a, signals A and B are indicated.
spectrum owing to magnetic field orientation of the MLV, there are significant differences with respect to their quadrupolar splittings. While the MLV exhibit one splitting of 22.9 kHz only, the SSV show two splittings of 28.5 kHz (signal A) and 22.7 kHz (signal B) of similar amplitude. Hence, the MLV splitting corresponds roughly to signal B of the SSV. This comparison between the two different systems and the similar intensities (cf. Figure 2b) of the separate SSV signals indicates that the presence of the solid support causes one monolayer leaflet of the SSV to adopt a higher molecular order (giving signal A) than that of the bilayers in MLV. This assignment of signals A and B of the SSV to the
two monolayer leaflets of the supported bilayer allows us to study the diffusion in the two layers separately in a suitable NMR relaxation experiment. A CPMG pulse sequence can act as a low pass filter for such diffusive motions and thus allow the determination of the diffusion constant, provided that the diameter of the spherical support is known.14,15 The results of a CPMG experiment on SSV of fluid DPPC-d8 (T ) 55 °C) for increasing time t ) nτ, where n is the number of echos and τ is the time between the 180° refocusing pulses are shown in Figure 2a for the case of τ ) 20 µs. To obtain the intensities of signals A and B as a function of time, we have numerically deconvoluted the CPMG spectra using the well-known de-Pakeing algorithm.13 The oriented spectra (Figure 2b) were then represented as overlapping Lorentzian lines, and the intensities of the separated peaks A and B were plotted semilogarithmically versus t as shown for the case of signal B in Figure 3a. This procedure must be considered as a first iteration since the de-Pakeing procedure does not explicitly consider the subtle changes of the spectrum with increasing t due to anisotropic T2 relaxation. The resulting deviations manifest themselves in the systematic baseline distortion of the de-Paked spectra. Within the limit of short t (t < 500 µs), the plot of the CPMG intensities vs t for different values of τ (Figure 3a) is exponential for both signals and the slope gives 1/T2CPMG(τ) for signal A and for signal B. According to the theory of transverse relaxation with lateral diffusion being the only slow motion mechanism and interpreting the relaxation rate of the de-Paked spectrum as the powderaveraged relaxation rate,16 this relaxation can be described by (T2CPMG)-1 ) [2M2rD/R2]τ2 + (T′2)-1 in the limit of τD . τM. Here M2r is the residual second moment of the 2H(15) Bloom, M.; Sternin, E. Biochemistry 1987, 26, 2101.
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Letters
Table 1. 2H-NMR Parameters and Lateral Diffusion Coefficient D for DPPC-d8 Bilayers on a Spherical Support of Silica (R ) 320 nm) at 55 °C signal A signal B
∆νQ (kHz)
b (1011 s-3)
M2 (109 s-2)
D (10-12 m2/s)
28.5 22.7
9.4 ( 0.2 11 ( 1.0
6.41 4.06
7.5 ( 1.0 13.8 ( 1.2
NMR line shape, D is the lateral diffusion coefficient of the lipids, R is the radius of the bead, τM is the NMR time scale (≈10-5 s for 2H-NMR), and τD ) R2/6D is the diffusion correlation time on a sphere of radius R. (T′2)-1 is the relaxation rate due to processes having correlation times τ′2, which are fast on the NMR time scale (τ′2 , τM). Hence, a plot of 1/T2CPMG vs τ2 should give a straight line with the slope b ) 2M2rD/R2. This is shown for both signals in Figure 3b. We obtain b ) 0.94 × 1012 s-3 (signal A) and 1.1 × 1012 s-3 (signal B), and thus with R ) 320 ( 20 nm and the second moments in the limit M2r ≈ M2 ) (2π∆νQ)2/5 (cf. Table 1) as calculated for each signal from the oriented spectra (Figure 2), a lateral diffusion constant D is obtained according to D ) bR2/(2Mr) as DA ) (7.5 ( 1.0) × 10-12 m2/s (signal A) and DB ) (13.8 ( 1.2) × 10-12 m2/s (signal B). These results are summarized in Table 1. 4. Discussion The results from Table 1 indicate a difference of D between the two monolayers. The value of DB is close to that measured by field gradient methods in DPPC multilayers (D ) (12 ( 1) × 10-12 m2/s) at the corresponding temperature (T ) 55 °C).17 We assign signal B to the outer (distal) monolayer which should be least affected by the presence of the solid surface. Consequently, the inner (proximal) monolayer facing the silica surface shows a ∼50% reduced value of D together with increased molecular order. Furthermore, the difference in D by a factor of 2 between the two monolayers shows clearly that the frictional coupling between them is not sufficiently strong to cancel out the effect of this asymmetric frictional drag arising from the silica surface. This result is in marked contrast to those obtained previously using the FRAP method for measuring D in both monolayers.11 There a D value for DMPC of D ) 11 × 10-12 m2/s was measured at 45 °C for both monolayers. This would roughly correspond to our value for the distal layer considering the temperature dependence of D,17 since we measured at 55 °C. We assume that differences (16) Bloom, M.; Morrison, C.; Sternin, E.; Thewalt, J. L. Spin-echoes and the dynamic properties of membranes. In Pulsed magnetic resonance: NMR, ESR and optics, a recognition of E. L. Hahn; Bagguley, D. M. S., Ed.; Clarendon Press: Oxford, 1992; p 274. (17) Karakatsanis, P.; Bayerl, T. M. Submitted for publication in Phys. Rev. E.
between the experimental setups may account for this discrepancy. First, the surface roughness of the planar glass substrate used for FRAP studies is certainly higher than that of the silica beads.3 This in turn leads to the expectation of a higher thickness of the water film and thus a reduced friction between proximal monolayer and water film for the FRAP experiment. Indeed, the authors of the FRAP study estimated the thickness of their water film between 1 and 50 nm while for silica beads as support this thickness does not exceed 3 nm.4,8 Another reason might be the fact that for the FRAP experiment about 20% of the fluorescence was not recovered (R. Merkel, personal communication). One might speculate that this loss corresponds to those regions of the supported bilayer which were virtually immobilized (on the FRAP time scale) due to a direct contact with the molecular rough glass surface. Then those immobilized regions of the proximal layer would not contribute to the measured recovery curve and diffusion would appear more uniform over both layers. Another contribution to the frictional drag between proximal monolayer and solid surface could arise from direct monolayer-surface contact by collective surface undulations of the bilayer. Although most of these collective modes are overdamped, some high-frequency and low-amplitude (less than 10 Å) collective motions still persist18 and may lead to temporary close contact between proximal layer and silica surface. On the NMR time scale, this may result in a lower average D value for the proximal monolayer. Considering the smoothness of the silica beads compared to the spikiness of a glass surface on a molecular scale, the occurrence of such undulations seems more likely for the SSV studied in this work. 5. Conclusion Our results show that the presence of a smooth solid surface of silica at close distance (3 nm and less) affects significantly the lipid lateral diffusion in the proximal monolayer of a single, fluid DPPC bilayer despite the presence of a lubricating water film. In contrast, the outer monolayer is not affected, thus indicating a surprisingly low frictional coupling between the two leaflets. Acknowledgment. We are indebted to Professor Myer Bloom (Vancouver) for many helpful discussions and for reading the manuscript. LA9712810 (18) Pfeiffer, W.; Ko¨nig, S.; Legrand, J. F.; Bayerl, T. M.; Richter, D.; Sackmann, E. Europhys. Lett. 1993, 23, 457.
