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Polymer Cushions in Supported Phospholipid Bilayers Reduce Significantly the Frictional Drag between Bilayer and Solid Surface Johannes Schmitt,† Birgit Danner,‡ and Thomas M. Bayerl*,‡ Nimbus Biotechnologie, Karl-Heine -Strasse 99, D-04229 Leipzig, and Universita¨ t Wu¨ rzburg, Physikalisches Institut EP-5, D-97074 Wu¨ rzburg, Germany Received September 6, 2000. In Final Form: October 13, 2000 The reduction of fluid lecithin bilayer-substrate coupling by the presence of a hydrophilic polymer cushion in spherical supported bilayers of dipalmitoylphosphatidylcholine (DPPC) has been studied by measuring the lecithin molecular order and lateral diffusion using a deuterium NMR relaxation method. We obtained for both monolayers of the supported bilayer a lateral diffusion coefficient (D ) 13.5 × 10-12 m2/s at 55 °C) similar to that measured previously in the outer monolayer of a supported bilayer on a bare silica surface, indicating that the presence of the polymer cushion can indeed largely eliminate the slow down effect of the solid surface on the bilayer dynamics. Moreover, we found that the polymer cushion rendered the molecular order of the lipids identical to that measured for multilamellar bilayer vesicles (MLV). A comparison of the measured D values with those obtained for oriented planar multilayer stacks of DPPC strongly suggests that lateral diffusion in these systems is still a factor of 2 faster than that of single bilayers on a support.
Introduction Phospholipid bilayers on a solid support can provide the ultrasoft environment for the functional immobilization of integral membrane proteins, a key step in the elucidation of protein-lipid interaction mechanisms and in the study of protein-protein binding by surface sensitive physical techniques.1-4 To prevent activity losses of membrane-spanning proteins owing to contacts between the proteins and the (mostly inorganic) support surface, polymer cushions (surface grafted films of hydrophilic polymers) are often employed as a spacer between the bilayer and its solid support. Detailed knowledge of the structure and dynamics of polymer-modified supported bilayer systems is of paramount importance for the successful reconstitution of such proteins and for the proper function of biosensors derived from such composite structures. Supported bilayers without polymer cushions have been demonstrated to lack essential properties for integral membrane protein immobilization: The gap between bilayer and support is not more than 2 nm,5,6 insufficient to fit the hydrophilic domains of most membrane-spanning proteins in their natural conformation. As a consequence of this closeness to the solid surface and the resulting structuring of the trapped water molecules,7 the molecular order and dynamics of the bilayer are altered compared to those of a free bilayer.5,8,9 In particular, the * To whom correspondence should be addressed. Phone: 49931-888-5863. Fax: 49-931-888-5851. E-mail:
[email protected]. † Nimbus Biotechnologie. ‡ Universita ¨ t Wu¨rzburg. (1) Sackmann, E.; Tanaka, M. TIBTECH 2000, 18, 57-64. (2) Sackmann, E. Science 1996, 271, 43-48. (3) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1997, 35, 114773-14781. (4) Lindholm-Sethson, B.; Carrasco-Gonzalez, J. Langmuir 1998, 14, 6705-6708. (5) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 59, 289294. (6) Bayerl, T. M.; Bloom, M. Biophys. J. 1990, 58, 357-262. (7) Ko¨nig, S.; Sackmann, E.; Richter, D.; Zorn, R.; Carlile, C.; Bayerl, T. M. J. Chem. Phys. 1994, 100, 3307-3316.
lipid diffusion is hampered in the monolayer facing the solid surface, resulting in an asymmetric lateral diffusion between the two monolayers.10 Another consequence of the close proximity of bilayer and support is the creation of tension owing to changes in the molecular area of the lipid at the phase transition, which manifests itself by a shift of the phase transition temperature of the supported bilayer.11 Polymer cushions between bilayer and support should, at least in part, prevent these alterations of bilayer properties due to the increase of the gap width and due to the biocompatible surrounding created by the hydrophilic polymers. Here we report a direct comparison between bilayers on spherical solid supports regarding the effect of the polymer cushion on the molecular order and diffusion in the bilayer. As in the previous study of diffusion in supported bilayers without a polymer cushion,10 we have employed an NMR relaxation method which allowed the simultaneous measurement of the lateral diffusion in the two monolayer leaflets of a supported bilayer separately over rather short distances (200-300 nm) and without relying on bulky molecular labels. Experimental Section Solid-supported DPPC-d8 bilayers immobilized on bare silica beads (solid-supported vesicles, SSV), TRANSIL-DPPC-d8, and those immobilized on polymer-modified silica (PM-SSV), TRANSIL-DPPC-d8-IMMO, were obtained from NIMBUS Biotechnologie GmbH (Leipzig, Germany). The spherical support of both bilayer systems consisted of monodisperse nonporous silica beads with a radius of R ) 320 ( 20 nm. The dipalmitoylphosphatidylcholine used was selectively deuterated at the 7,7 and 8,8 positions of the two palmitoyl chains (DPPC-d8). TRANSILDPPC-d8-IMMO was produced by a proprietary multilayer coating procedure of the spherical silica support with the DPPCd8 bilayer tightly adsorbed to the top hydrophilic polymer layer. (8) Podornik, R.; Parsegian, V. A. Biophys. J. 1997, 72, 942-952. (9) Dolainsky, C.; Mo¨ps, A.; Bayerl, T. M. J. Chem. Phys. 1993, 98, 1712-1720. (10) Hetzer, M.; Heinz, S.; Grage, S.; Bayerl, T. M. Langmuir 1998, 14, 982-984. (11) Naumann, C.; Brumm, T.; Bayerl, T. M. Biophys. J. 1992, 63, 1314-1319.
10.1021/la001275v CCC: $20.00 © 2001 American Chemical Society Published on Web 12/09/2000
Polymer Cushions in Supported Phospholipid Bilayers
Figure 1. 2H NMR spectra of DPPC-d8 at 55 °C as multilamellar vesicles (top spectrum), as single bilayers on a spherical solid support of silica (SSV, middle spectrum), and as single bilayers on polymer-modified silica (PM-SSV, bottom spectrum). Vertical dashes indicate the identical quadrupolar splittings of MLV and PM-SSV. For NMR measurements, the samples were dispersed in deuterium-depleted water at a lipid concentration of 12 mg/mL. Multilamellar vesicles (MLV) were prepared by swelling the lipid (DPPC-d8 from Avanti Polar Lipids, Alabaster, AL) in deuteriumdepleted water for 1 h at 50 °C under gentle vortexing. The lipid concentration of the MLV sample was 20 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 scheme were used for these experiments. The CPMG echo intensities were determined from the 0th moment of the CPMG spectra. All other parameters of the measurement and methods of data analysis were described in detail previously.12 Differential scanning calorimetry (DSC) measurements of the samples were performed using a Hart calorimeter (Hart Scientific, UT) at a scan rate of 15 °C/h. The samples were dispersed in water at a concentration of 3-5 mg of lipid per milliliter.
Results We studied three types of spherical phospholipid bilayers: free multilamellar vesicles (MLV), a single bilayer on silica beads (solid-supported vesicles, SSV), and a single bilayer on polymer-modified silica beads (PMSSV). In a first set of experiments, we compared the phase transition temperature Tm of DPPC-d8 for MLV and for PM-SSV as measured by DSC. Both systems gave identical transition temperatures of Tm ) 40.5 ( 0.2 °C, but the width of the transition was a factor of 3 larger for the PM-SSV. It has been previously shown that the Tm of DPPC in SSV (beads without a polymer cushion) is reduced by about 2 °C compared to that of MLV, owing to the tight coupling of the bilayer with the spherical solid support. This creates a lateral tension in the bilayer due to the 5-10% lipid molecular area shrinkage at the transition to the gel phase. For PM-SSV, the effective decoupling of the bilayer from the support by a soft, hydrophilic, and flexible polymer cushion seems to prevent the creation of tension, leaving the Tm at the same value as that for the MLV. Figure 1 shows the 2H NMR spectra of the three systems studied at 55 °C (fluid phase). Three obvious differences were observed: (1) The MLV sample exhibited a line shape distortion of the spectrum owing to the magnetic field orientation while the supported systems (SSV and PMSSV) did not show any deviation from a spherical (12) Ko¨chy, T.; Bayerl, T. M. Phys. Rev. E 1993, 47, 2109-2116.
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distribution of molecular director axes. (2) The SSV exhibited two quadrupolar splittings of νQA ) 24.5 kHz and of νQB ) 20.5 kHz of similar amplitude while both PM-SSV and MLV showed just a single splitting of νQ ) 21.6 kHz. It was previously shown that the two splittings of the SSV spectrum arise from an asymmetry in the molecular order between the two monolayer leaflets of the supported bilayer with the higher splitting (νQA) belonging to the inner monolayer facing the silica support.10 (3) The absence of any double splitting feature for the PM-SSV sample and a νQ identical to that of the MLV sample (a free bilayer system without any interference from solid surfaces) lets us assume that the polymer modification of the solid support causes a softening of the solid/liquid interface in such a way that the molecular order asymmetry as observed for SSV samples is rendered undetectable in PM-SSV. A critical test of the above surmise is the measurement of the lateral diffusion constant D of the phospholipids. We have previously shown that, for SSV, the value of D for the inner monolayer is about 50% lower than that of the outer layer due to the close proximity of the former to the solid surface.10 If the polymer cushion between bilayer and solid support caused indeed a dynamic decoupling of lipid diffusivity from the solid surface, then D of PM-SSV should be at least equal to that measured in the outer monolayer of the SSV. As in the previous SSV study,10 a Carr-PurcellMeiboom-Gill (CPMG) pulse sequence has been used to measure DPPC-d8 lateral diffusion of PM-SSV. This sequence can act as a low pass filter for such diffusive motions and thus allows the determination of the diffusion constant, provided that the diameter of the spherical support is known.12,13 The results of a CPMG experiment on PM-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 2 for the case of τ ) 20 µs. Within the limit of short t (t < 500 µs), the semilogarithmic plot of the CPMG intensities versus t ) nτ for different values of τ (Figure 2A) was linear for all values of τ, and the slope of the linear fits gave 1/T2CPMG(τ). According to the theory of transverse relaxation with lateral diffusion being the dominant slow motion mechanism,13 this relaxation can be described by (T2CPMG)-1 ) [2M2rD/R2]τ2 + (T2′)-1 in the limit of τD . τM. Here M2r is the residual second moment of the 2H 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 τ ) R2/6D is the diffusion correlation time D on a sphere of radius R. (T2′)-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 versus τ2 should give a straight line with the slope b ) 2M2rD/R2 (Figure 2B). We obtained b ) 11.48 × 1011 s-3, and thus with R ) 320 ( 20 nm and the second moments in the limit M2r ≈ M2 ) (2πνQ)2/5 ) 4.55 × 109 s-2, a lateral diffusion constant D is obtained according to D ) bR2/ (2Mr) as DlPM-SSV ) (13.5 ( 1.5) × 10-12 m2/s at 55 °C. These results are summarized in Table 1, along with those obtained for SSV and MLV samples previously. Discussion From Table 1 it is obvious that MLV samples exhibit the highest Dl while that of both types of spherical (13) Bloom, M.; Morrison, C.; Sternin, E.; Thewalt, J. L. In Pulsed magnetic resonance: NMR, ESR and optics, a recognition of E. L. Hahn; Bagguley, D. M. S., Ed.; Clarendon Press: Oxford, 1992; pp 274-316.
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Table 1.
2H
Schmitt et al.
NMR Parameters and Lateral Diffusion Coefficient D for a Single DPPC-d8 Bilayer on a Spherical Support of Polymer-Modified Silica (PM-SSV) and for Multilamellar Vesicles (MLV) at 55˚Ca
sample PM-SSV SSV MLV planar multilayers
inner monolayer outer monolayer
νQ
b
M2
D
kHz
× 1011 s-3
× 109 s-2
× 10-12 m2/s
21.6 24.5 20.5 21.6
11.5 ( 1.0 9.4 ( 0.4 11 ( 1.0
4.55 6.41 4.06
13.5 ( 1.5 7.5 ( 1.0 13.8 ( 1.2 21.5 ( 1
a
Also shown are the values obtained previously for a single DPPC-d8 bilayer on bare silica and for (planar) DPPC multilayers14 at the same temperature. The diameter of the spherical support was 320 ( 20 nm for both SSV and PM-SSV.
Figure 2. CPMG relaxation measurements for PM-SSV at 55 °C. (A) Semilogarithmic plot of the CPMG signal intensity versus time t (t ) 500 µs) for different values of τ ) 16, 20, 30, 35, and 40 µs as indicated. The slope of the linear fits (dotted lines) gave 1/T2CPMG(τ). (B) Semilogarithmic plot of 1/T2CPMG(τ) versus τ2. The slope of the linear fit (full line) gave b, from which Dl was calculated.
supported bilayer samples is 30% lower. It is interesting to note that the value of D in the outer monolayer of the SSV agrees very well with that of the PM-SSV. This strongly supports the above surmise that the polymer cushion of the PM-SSV can indeed soften the solid/fluid interface in a way that the lateral diffusion of the lipids is no longer slowed for the inner monolayer by the proximity of a solid surface. Hence, the PM-SSV system shows diffusive properties and a molecular order closer to that of a free bilayer. One should note that Table 1 indicates that for MLV and PM-SSV (both are curved systems) the molecular order as expressed by the quadrupolar splitting is exactly the same. Nevertheless, the PM-SSV lateral diffusion is still significantly slower than
(SSV)10
that measured for planar multilayers of the same lipid (Table 1) at this temperature. Since both methods used for measuring D in spherical and planar systems (Table 1) have demonstrated in the past their high precision in the determination of D in lipids, the slower diffusion in SSV and PM-SSV compared to MLV must arise from the geometrical or morphological differences between the samples. Since the order parameters of the PM-SSV and of the intrinsically tension free MLV are identical (Figure 1), we assume that the curvature does not account for the discrepancy in the D values. Thus, it seems that the multilamellarity of the planar sample must be the reason for the faster lipid diffusion. The average thickness of the water layer in such fully hydrated samples is 25 Å,15 which is higher than that in SSV (10-15 Å6), while in PM-SSV the separation between the hydrophilic polymer cushion and the bilayer can even be less than 10 Å. This closeness to the surface (silica or polymer) may contribute to a general slowing of lateral diffusion in the single-bilayer systems. Furthermore, membrane undulations are present in planar multilamellar systems15,16 but not in SSV.9 Diffusive damping of collective modes is a transverse relaxation process and thus may contribute to the measurement of D in multilamellar samples. As a result, the D value calculated from a spin-echo experiment in multilamellar systems may apparently be larger than that in single supported bilayers where this contribution is not detectable owing to the complete overdamping of collective modes by the solid surface. Conclusion We have shown that the chemical modification of a silica surface by a polymer layer can drastically reduce the effect of the solid surface on the phase behavior, the molecular order, and the lipid diffusion of a supported bilayer. The polymer-modified supported system exhibits a molecular order and a phase transition temperature identical to those of bilayers in multilamellar vesicles. This reduction of solid surface disturbance can provide the key for the functional coupling and reconstitution of peptides and proteins in supported bilayers. LA001275V (14) Karakatsanis, P.; Bayerl, T. M. Phys. Rev. E 1996, 54, 17851790. (15) Vogel, M.; Munster, C.; Fenzl, W.; Salditt, T. Phys. Rev. Lett. 2000, 84, 390-393. (16) Pfeiffer, W.; Ko¨nig, S.; Legrand, J. F.; Bayerl, T. M.; Richter, D.; Sackmann, E. Europhys. Lett. 1993, 23, 457-462.
