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Supported Hybrid Bilayer Membranes as Rugged Cell Membrane Mimics Anne L. Plant Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8311 Received November 30, 1998. In Final Form: March 26, 1999 Supported planar lipid bilayers based on alkanethiol-tethering chemistry are becoming increasingly important biomimetic materials. Hybrid bilayers containing thiol-derivatized alkane moieties plus natural lipids provide a biomimetic matrix that permits the successful reconstitution of membrane protein activity. The hybrid membrane is self-assembled and sufficiently rugged to be of practical use in research and in commercial sensing applications. The coupling of the bilayer to a metal support allows a wide range of analytical techniques to be applied to this model membrane. This article reviews some of the fundamental studies of the formation, structure, and response of hybrid bilayer membranes.
Introduction The Cell Membrane and Need for Biomimetic Models. The boundaries of biological cells and organelles are defined by membranes that are as dynamic and complex as the cell itself. The cell membrane is an array of lipids, proteins, and carbohydrates that carries out functions that include molecularly specific transport, receptor binding, enzymatic activity, and control of cell-cell interactions. Signal transduction of binding events at the cell surface result in cascades of second messengers inside of the cell that ultimately control intracellular processes such as protein synthesis and cell replication. Studying the structure and function of the membrane-bound proteins responsible for these activities presents special challenges, since, in general, these proteins must remain in a lipid matrix to retain their native structure and activity. Isolation and purification of these proteins are far from routine. Achieving high-resolution structural information about membrane proteins is a serious current challenge. Simplified models of cell membranes have been the subjects of intense study. The primary components of model membranes are amphiphilic molecules, predominately phospholipids, which self-assemble in water in thermodynamically controlled aggregate structures.1 In phospholipid bilayers, the hydrophobic alkane chains are sequestered from water, and polar moieties are in contact with water. The individual lipid molecules can be dynamic in their molecular order, their location in the plane of the bilayer, and their equilibration with aqueous-phase monomers. The aggregate bilayer structure is long-lived and is chemically and electrically insulating. Many kinds of model membranes have been used in research, including lipid vesicles or liposomes, black lipid membranes (BLMs, also known as bilayer lipid membranes), Langmuir-Blodgett layers, and tethered and nontethered supported planar bilayers.2,3 All have advantages and disadvantages with respect to ease of use and accuracy of mimicking “the real thing”. Phospholipid vesicles and liposomes have important applications in drug (1) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley and Sons: New York, 1973. (2) Jain, M. K.; Wagner, R. C. Introduction to Biological Membranes; John Wiley and Sons: New York, 1980. (3) Sackman, E. Science 1996, 271, 43-48.
10.1021/la981662t
delivery4 and as labels in analytical applications, but few other applications for model membranes have been developed. One could imagine harnessing these insulating membranes containing receptor, enzyme, or channel proteins for applications in sensors, pharmaceutical screening, chemical synthesis, bioremediation, and separations. The technical impediments to this goal include ease of formation of the membrane, long-term integrity of the structure, and reconstitution of functional proteins in their native conformation. We have been examining the use of alkanethiol selfassembled monolayer technology as a starting point for forming stabilized, rugged, biomimetic bilayer membranes. Hybrid bilayer membranes (HBMs) represent a means of stabilizing a lipid bilayer while preserving its dynamic nature. Results from our lab and others suggest that such constructs provide a way of organizing membrane proteins in a native membrane-like environment. In addition, membranes tethered to metal surfaces are electrochemically and optically addressable while providing a physiologically compatible coating. This article reviews some of the progress that has been made in HBM structural characterization and demonstration of application, and discusses the future of this technology. Advantages of Hybrid Bilayer Membranes The use of phospholipid vesicles to spontaneously coat a covalently tethered hydrophobic layer on metal with a monolayer of solvent-free lipid5-7 has opened up a new era in biomimetic model membranes. We refer to alkanethiol/phospholipid bilayers as “hybrids” because they consist of both natural and synthetic components (Figure 1). The use of alkanethiols provides a distinct advantage over that of other planar model membranes. The alkanethiols can form a complete hydrophobic layer at metal surfaces8 and provide the driving force for the formation (4) Liposome Technology. Interctions of Liposomes with the Biological Milieu, 2nd ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1992; Vol. III. (5) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667-1671. (6) Lang, H.; Duschl, C.; Gratzel, M.; Vogel, H. Thin Solid Films 1992, 210/211, 818-821. (7) Plant, A. L. Langmuir 1993, 9, 2764-2767. (8) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 1-4483.
This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society Published on Web 06/17/1999
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reflectivity, ellipsometry, nonlinear optical spectroscopy, reflection-absorption infrared spectroscopy, and even vacuum techniques. The applicability of such a wide range of analytical techniques to biomimetic membranes opens up exciting new avenues for studying the complex structure and function of biological membranes. Formation of Hybrid Bilayer Membranes
Figure 1. Alkanethiol/phospholipid hybrid bilayer membrane. The orientation of the layer of phospholipid that assembles at the akanethiol monolayer maximizes hydrophobic interactions.
of a complete bilayer. The covalent association with the surface is insensitive to changes in buffer, pH, ionic strength, or lipid composition. Fabrication is easy, since both the monolayer preparation and the formation of the bilayer are self-assembly processes. HBMs can be kept intact and studied for months; they are predicted to have significantly more mechanical stability than suspended BLMs.9 Furthermore, because the tethered HBM is formed at a surface, many techniques that have not been generally applied to biological membranes are now accessible. The use of a metal support, such as gold, permits the application of electrochemical techniques for examining the insulating character of the lipid layers6,7,10-13 and for assessing the activity of membrane protein pores,7,11 lipase10 and redox enzymes,14-16 proton translocators,17-19 and ionophores.20-22 Also, the metal layer allows the use of surface plasmon resonance to examine the formation of these biomimetic membranes5,23-26 and the association of solution-phase molecules to surface membrane-bound receptors.27-29 The planarity and stability of these bilayers also facilitate the use of atomic force microscopy, neutron (9) Florin, E.-L.; Gaub, H. E. Biophys. J. 1993, 64, 375-383. (10) Stelzle, M.; Weissmuller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (11) Plant, A. L.; Guegeutchkeri, M.; Yap, W. Biophys. J. 1994, 67, 126-1133. (12) Steinem, C.; Janshoff, A.; Ulrich, W.-P.; Sieber, M.; Galla, H.-J. Biochim. Biophys. Acta 1996, 1279, 169-180. (13) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenha¨usser, A. Langmuir 1997, 13, 7085-7091. (14) Kinnear, K. T; Monbouquette, H. G. Langmuir, 1993, 9, 22552257. (15) Torchut, E.; Bourdillon, C.; Laval, J. Biosens. Bioelectron. 1994, 9, 719-723. (16) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. Langmuir 1998, 14, 2467-2475. (17) Seifert, K.; Fendler, K.; Bamberg, E. Biophys. J. 1993, 64, 384391. (18) Nauman, R.; Jonczyk, A.; Hampel, C.; Ringsdorf, H.; Knoll, W.; Bunjes, N.; Gra¨ber, P. Biochem. Bioenerg. 1997, 42, 241-247. (19) Steinem, C.; Janshoff, A.; Hohn, F.; Sieber, M.; Galla, H. J. Chem. Phys. Lipids 1997, 89, 141-152. (20) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580583. (21) Jenkins, A. T. A.; Bushby, R. J.; Boden, N.; Evans, S. D.; Knowles, P. F.; Liu, Q.; Miles, R. E.; and Ogier, S. D. Langmuir 1998, 14, 46754678. (22) Raguse, B.; Graach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 648659. (23) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (24) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Gra¨ber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 6188-6194. (25) Hubbard, J. B.; Silin, V.; Plant, A. L. Biophys. Chem., in press. (26) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757. (27) Plant, A. L.; Brigham-Burke, M.; Petralli, E. C.; O’Shannessy, D. Anal. Biochem. 1995, 226, 342-348.
When phospholipid vesicles in aqueous media are exposed to the hydrophobic alkanethiol-coated surface, lipid molecules spontaneously assemble into a second layer over the alkanethiol monolayer. The process of addition of lipid to the monolayer can be followed by decreases in electrical capacitance with time.27 The driving force for the self-assembly of phospholipids at an alkanethiol monolayer is presumably the hydrophobic effect, assuming that the result of addition of the phospholipid layer is the reduction of the free energy of the alkanethiol/water interface. Direct evidence for this was pursued by examining the effect of addition of a layer of phospholipid to the alkanethiol monolayer using surface enhanced Raman spectroscopy (SERS) and reflection-absorption infrared spectroscopy (RAIRS).30 Both techniques indicated that changes in the alkanethiol monolayer are subtle. SERS was performed on monolayers in contact with water during bilayer formation. These measurements allowed assessment of the effect of the change in the microenvironment of the alkanethiols as water was replaced with the hydrophobic phospholipid layer. SERS indicated that the short chain alkanethiols, which are relatively disordered, are more affected by the addition of a layer of phospholipid than the longer chain thiols. Hexanethiol showed increases in the trans/gauche ratios of conformers of C-S, C-C, and terminal CH3 groups, indicating an increase in intramolecular order. We also observed changes in the C-H stretching region that indicate an increase in intermolecular order and are analogous to changes that accompany a decrease in temperature30 (Figure 2). Longer chain alkanethiols showed little structural change due to the addition of phospholipid. RAIRS measurements compared alkanethiol monolayers in air with alkanethiols as components of HBMs in air, so the change in the environment of the alkanethiol layer is expected to be less drastic than that when the films are examined in water. Small changes in peak intensities were observed with RAIRS in the C-H stretching region. Increases in the intensities of the CH2 peaks were more prominent as the alkanethiol chain length increased, while increases in the CH3 peak intensities were not. These changes are also consistent with changes in alkanethiols induced by low temperatures.31 Thus, both SERS and RAIRS results suggest that phospholipid addition to the alkanethiol layer has an effect similar to that of reducing the temperature. This is consistent with the process being thermodynamically favorable and provides direct evidence of the role of the free energy of the surface in bilayer formation. It is also significant that these spectral changes are small, particularly for long chain thiols, indicating that the structure of the alkanethiol monolayer does not change significantly as a result of becoming part of a bilayer. This simplifies (28) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361-1369. (29) Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H. Biochemistry 1998, 37, 507-522. (30) Meuse, C. W.; Niaura, G.; Lewis, M. L.; Plant, A. L. Langmuir 1998, 14, 1604. (31) Nuzzo, R. G.; Korenic, E. M.; Dubois, L. H. J. Chem. Phys. 1990, 93, 767.
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Figure 2. SERS spectrum of a hexanethiol monolayer as a component of HBMs: (A) hexanethiol monolayer on roughened Ag before (s) and after (‚‚‚) addition of deuterated DMPC; (B) hexanethiol monolayer at 343 K (s) and 278 K (‚‚‚). Parts A and B are offset from one another for clarity.
the optical and electrical characterization of the phospholipid portion of the bilayer, because the underlying monolayer can be treated as a reasonably good reference layer. It is relevant to question what the driving force is for the self-assembly of the lipid layer at the alkanethiol monolayer surface. Since the lipid vesicle is a long-lived metastable structure, the conversion of a bilayer vesicle to a surface monolayer might be expected to involve a significant activation energy barrier. From a practical point of view, understanding the thermodynamics of the rearrangement might provide strategies for enhancing the rate of bilayer formation or the stability of the bilayer structure. The first step in understanding this process is to identify the rate-determining step in HBM formation. There are at least two potential mechanisms by which a bilayer could form. The addition of a layer of lipid to the hydrophobic monolayer could occur via a vesicle-dependent process as has been suggested,32 or formation could occur by individual monomer phospholipids transferring from the vesicles to the aqueous phase and from there to the hydrophobic surface. If the process occurs by aqueous phase transfer, prediction of the final composition of the HBM would be difficult; the lipid layer of the HBM may not resemble the composition of the mixed component vesicle population. We used surface plasmon resonance (SPR) to study the mechanism of bilayer formation. Changes in the refractive index of the metal interface result in changes in the angular dependence of the reflection of an incident beam of light from the surface. With SPR, additions and losses of material from the surface can be monitored directly (32) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316.
Figure 3. Kinetics of addition of phospholipid to the alkanethiol surface measured for different concentrations of vesicles (in mg mL-1) by surface plasmon resonance. All data were fit with a model which accounted for diffusion plus a surface reorganization kinetic constant.
without the need for secondary labels. We examined the rate of bilayer formation as a function of the concentration of phospholipid vesicles. The time-dependent increase in optical thickness (a combination of thickness and refractive index) indicated addition of lipid to the monolayer surface25 (Figure 3). The rate of bilayer formation increased with increasing vesicle concentration. The time-dependent surface coverage Γ(t) can be described by Fickian diffusion to an imperfectly absorbing (partially reflecting) surface whose active sites are blocked by previously adsorbed lipid. At Γm, the maximum surface coverage of phospholipid, is reached.
Γ(t) )
(
∫0tdτ 1 -
)
Γ(τ) F (τ) Γm D
The diffusive flux to the surface, FD, is given by
FD ) KC0 exp
( ) (
)
K2t K erfc 1/2t1/2 D D
where C0 is the bulk concentration of the absorbing species (vesicles or monomer phospholipid), D is the bulk diffusion coefficient of the vesicles or monomer phospholipid, K is a surface reorganization rate constant or inverse reflection coefficient (in cm s-1), and erfc indicates the complementary error function. In this model, the kinetic constant K reflects a surface reaction with linear dimensions of centimeter per second. We speculate that this constant may be the product of a surface diffusion constant, with
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units of square centimeter per second, and a reorganization constant, with units of inverse second. The equation derived from the above expressions adequately fits the kinetic data over the entire concentration range25 (Figure 3), but limiting expressions of the model could also explain the data. At the limit of large values of K2/D, the kinetics are accounted for by diffusion alone. The expression for this limiting case fit the data well at high concentrations of vesicles. The diffusion coefficient corresponded to the diffusion coefficient of the phospholipid vesicles, which was measured by quasielastic light scattering. This limit is consistent with an absorbing boundary condition: vesicles that approach the surface are rapidly converted to an HBM, resulting in depletion of vesicles in the solution near the surface. At the limit of very small values of K2/D, the kinetics are independent of diffusion and are controlled by K, the reorganization rate constant. This limit described the data for the lower concentrations of vesicles. It is consistent with the existence of a reflecting boundary: vesicles can approach the surface and then leave without interacting with it, thereby maintaining a constant concentration of vesicles near the surface. Explicit consideration of two competing species with different diffusion coefficients (i.e., one for vesicles and one for monomer lipid) had little effect on the goodness of fit or the fitted parameters. Thus lipid monomers apparently do not play a direct role in the process of bilayer formation. The data indicate that at high concentrations the process of HBM formation is clearly vesicle-dependent. This knowledge simplifies the prediction of the final composition of HBMs prepared from vesicles. The diffusion limit of this model is also valid when cell membranes are used in place of phospholipid vesicles. Osmotic lysis was used to prepare red blood cell membranes (RBC ghosts) which consist only of the plasma membrane and are devoid of cellular contents. These are much larger structures (2-3 µm in diameter as determined by quasielastic light scattering) than the phospholipid vesicles. The RBC ghosts form HBMs at the alkanethiol monolayer surface by adding to the surface the equivalent of a 3 nm layer with a refractive index of 1.4.33 This thickness is consistent with the thickness of half a cell membrane bilayer. The kinetics of this process are diffusion-limited but with a slower rate than that observed for the vesicles, as is consistent with the slower diffusion coefficient for the larger particles. Similar data have since been observed in our lab for formation of cell membrane hybrid layers from COS cell membranes and 293T cell membranes. Structure and Function of Hybrid Bilayer Membranes How well do HBMs mimic biological membranes? Structurally, the phospholipid portion of HBMs is consistent with that of phospholipid bilayers. HBMs formed from well-organized alkanethiol monolayers, including hexane-, decane-, and octadecanethiol,9 demonstrate a stable and relatively uncomplicated impedance response that can be modeled simply as a parallel resistor/capacitor (RC) circuit l1 (Figure 4). Tethered bilayers containing more complicated underlying monolayers demonstrate a more complicated impedance response,22 but providing that the response can be modeled with only a few circuit elements, a great deal of structural and functional information can be derived from impedance (33) Rao, N. M.; Plant, A. L.; Silin, V.; Wight, S.; Hui, S. W. Biophys. J. 1997, 73, 3066-3077.
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Figure 4. Impedance response of an HBM composed of dioleoylphosphatidylcholine/decanethiol. The shape of the response is invariant over a range of applied electrical potentials, -0.2 V (b), -0.4 V (0), and -0.55 V (O), and can be fit with a simple equivalent circuit model. Capacitance values allow estimation of the bilayer thickness. Inset: the complex plane plot (real versus imaginary component of the impedance) of the data collected at -0.55 V demonstrates that the response appears to be the result of a single kinetic component, indicating a homogeneous dielectric layer.
analysis. Only the capacitance of the acyl portion of the phospholipid contributes significantly to the impedance of the lipid layer, since the capacitance of the hydrated polar headgroup region is relatively large. The impedance response from HBMs indicates a homogeneous addition of a layer of phospholipid to the alkanethiol layer. The impedance as a function of frequency permits the determination of the specific capacitance (i.e., normalized to electrode area) of the phospholipid layer, Cm-PL. This is estimated from the difference in capacitance of the original monolayer and the final bilayer:
1 1 1 ) Cm-PL Cm-bilayer Cm-monolayer With the capacitance value of the new layer and an estimate of its dielectric constant κ, one can estimate the thickness d of the lipid layer:
Cm-PL ) 0κ/d where 0 is the permittivity of free space. This analysis assumes that the alkanethiol monolayer does not change significantly with the addition of the phospholipid layer; vibrational spectroscopy confirms this assumption, as was discussed earlier. It is significant that the HBMs can be prepared in the absence of organic solvent, since most capacitance data available on BLMs reflect the effect of decane, the solvent used in their formation. Examination of the effect of the phospholipid chain length shows a linear increase in impedance with chain length and provides an estimate of the dielectric constant of the phospholipid layer of 2.7,11 compared to the accepted value of 2.1 for alkanes such as decane.
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above the phase-transition temperature of DPPC. At the higher temperature, where we expect DPPC to be in its liquid crystalline phase, the thickness of the hydrocarbon region of the lipid layer decreased by 0.1-0.2 nm relative to its thickness at 20 °C, and the area of the lipid molecules increased from 0.504 ( 0.016 to 0.608 ( 0.046 nm2. These observations are consistent with previously published values based on X-ray diffraction data from multi-bilayer systems. Some of our observations with HBMs are different from what has been reported with multi-bilayers. For example, we observe only a small decrease in the thickness of the lipid headgroup region (0.2-0.3 nm) at the higher temperature, instead of the difference of a factor of 2 that has been reported on the basis of X-ray data. We calculate that there are about 11 water molecules associated with each phospholipid molecule, which is consistent with recent X-ray data.36 Because HBMs are single bilayer membranes in contact with bulk water, our analysis eliminates the uncertainty that can be associated with knowing whether stacks of lipid bilayers are truly hydrated. Recent molecular dynamics simulation results37 indicate that the headgroups of HBMs and phospholipid bilayers are very similar. Both systems appear to have about 10 water molecules per headgroup in the liquid crystalline phase, but the simulations suggest that the gel-phase lipid has about half that number of water molecules associated per headgroup.
The structure of the phospholipid layer also has been examined in our laboratory by reflection-absorption infrared spectroscopy (RAIRS) and by neutron reflectivity. RAIRS of the dipalmitoylphosphatidylcholine (DPPC) layer of an HBM in air shows that it is highly ordered, with 95 ( 4% trans conformers, as indicated by analysis of the C-H wagging progression bands between 1100 and 1400 cm-1.30 Comparison of observed and simulated spectra34 indicate that the tilt of the phospholipid layer is approximately 34° from normal to the surface, consistent with that seen in phospholipid multi-bilayer systems. The phospholipid portion of HBMs also undergoes temperature-dependent phase transitions. We have confirmed the structure of HBMs and observed the effect of temperature on the thickness of HBMs using neutron reflectivity.34 Neutron reflectivity provides the opportunity for high-resolution characterization of the composition of these layers in the axis perpendicular to the surface. Recent advances in sample configuration, measurement cells, and instrumentation contribute to great sensitivity at the NIST Center for Neutron Research.35 We have examined HBMs out to wavevector transfer values (Q) of nearly 0.05 nm-1 and to reflectivities as low as 10-8 of incident radiation. We compared DPPC/octadecanethiol HBMs at 20 and 60 °C34 (Figure 5), which are below and
Protein Function in Hybrid Bilayer Membranes Demonstration that proteins can function in HBMs is essential for assessing the biomimetic character of these model membranes. A number of studies have demonstrated the activity of membrane proteins in thiol-tethered lipid bilayers. Purified Proteins in Tethered Bilayers. Several groups have demonstrated that diffusion of proteins and lipid components in the phospholipid layer of thiol-tethered bilayers allows reconstitution of multicomponent function. In biological membranes, lipid and protein components can diffuse laterally in the plane of the bilayer. Torchut et al.15 have shown that ubiquinone diffusion in alkanethiol/phospholipid bilayers occurs with a rate constant of 1.7 × 10-8 cm2 s-1, which is not very different from the rate constants measured for lipid diffusion in other model membranes, and that it transfers electrons with membrane-bound pyruvate oxidase. In a demonstration of the use of tethered bilayers in the design of sensors, the Cornell group have shown that gramacidin diffuses in the outer leaflet of the tethered membrane, allowing completion of a transmembrane ion channel with tethered gramacidin molecules.20 The Vogel group have demonstrated the reconstitution of functional rhodopsin, which, when activated with light, interacts with membrane-bound G-protein transducin, resulting in release of the G-protein subunits from the membrane.29 Solution-phase cytochrome c was shown to transfer electrons with cytochrome c oxidase in tethered membranes, and electrochemical evidence suggests that cytochrome c oxidase is able to diffuse transversely within the membrane toward the electrode during anodic scans.16 This study is an example of the potential usefulness of electrochemical techniques and tethered membranes to gain new insight into mechanisms of action of membrane proteins. Another protein that this approach can provide functional information about is the pore-forming peptide
(34) Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J. A.; Fu, J.; Connor, J. T.; Plant, A. L. Biophys. J. 1998, 74, 1388-1398. (35) Dura, J. A.; Richter, C. A.; Majkrzak, C. F,; Nguyen, N. V. Appl. Phys. Lett. 1998, 73, 2131-2133.
(36) Tristram-Nagle, S.; Zhang, R.; Suter, R. M.; Worthington, C. R.; Sun, W.-J.; Nagle, J. F. Biophys. J. 1993, 64, 1097. (37) Tarek, M.; Tu, K.; Klein, M. L.; Tobias, D. J. Biophys. J. 1999, 77.
Figure 5. (A) Log reflectivity versus Q (wavevector transfer) of an octadecanethiol/ DPPC HBM in D2O at 20 and 60 °C. The lines through the data represent the best model-dependent fits. (B) Neutron-scattering length density profile F as a function of distance from the surface Z from the model-dependent fits of the raw data.
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Figure 6. Before melittin is added to the HBM (9), reduction of Fe3 CN63- is greatly impeded. The addition of melittin creates a defect or pore and increases Fe3CN63- reduction by reducing the resistance to the flow of electrons through the membrane (b). The activity of the melittin lesion decreases with continuous cycling of electrical potential (after 5 min (0), 10 min (O), 20 min (*)). When the membrane is left at resting potential for 1 h (×), the melittin activity partially recovers.
melittin. Melittin is the toxin in bee venom and is a small protein of 26 amino acids. It is a model compound for a class of peptide membrane disrupters. Although soluble in water, monomers of melittin associate with membranes and subsequently aggregate into an oligomeric structure that is responsible for increasing the permeability of the membranes.38 Most of the studies of the activity of melittin have measured its effect on leakage of the aqueous contents from lipid vesicles. The size of the oligomeric complex and the orientations of monomer and oligomeric melittins in bilayers are still a source of controversy. We are currently examining the orientation and depth of penetration of melittin in HBMs by neutron reflectivity. In HBMs, melittin shows behavior that is consistent with observations of its activity in other model systems. Following the addition of melittin, voltammetry indicates an increased permeability of the HBM to aqueous redox molecules and thus an increase in potential-dependent current. Melittin activity as a pore or channel has been proposed to be potential-dependent.39 We have observed by electrochemistry with HBMs variable time-dependent behavior from melittin as a function of the number of potential scans (Figure 6). The effect of melittin in HBMs is blocked when melittin is added in a solution of high ionic strength,7 which has been attributed to the formation of an oligomeric complex in solution that cannot insert into membranes.38 We have shown with impedance analysis that the effect of melittin on electron transfer is the result of a decrease in the resistance of the bilayer and that the change in the capacitance is very small.11 Thus, the membrane structure is not destroyed, but it is less insulating. Using voltammetry, we can show that melittin produces an observable effect on the permeability of the membrane to ions within minutes of its addition to the HBM and that its effect on the membrane is persistent. Melittin appears to remain active in the membrane for days after removing excess peptide from solution. Such information cannot be obtained from measurements based on the transient leakage of lipid vesicle contents. A unique advantage of studying proteins in tethered membranes on metal surfaces is that it offers the possibility of applying electrical potential while simultaneously employing optical or other techniques to probe
resulting changes in protein structure or function. We have examined cytochrome c adsorbed to HBMs containing 20% negatively charged lipid. Using surface-enhanced resonance Raman spectroscopy (SERRS), the structure of the heme moiety was monitored as the iron center was reduced (Figure 7). The heme moiety of the HBMassociated cytochrome c retains a native in-plane conformation, unlike the case for cytochrome c adsorbed to a bare metal electrode.40 Cell Membrane Hybrids. As described above, RBC membranes behave in a similar fashion to that of phospholipid vesicles when exposed to alkanethiol monolayers: a monolayer of cell membrane appears to associate strongly with the surface to form a bilayer.33 Atomic force microscopy, ellipsometry, and SPR all suggest that the cell membrane adds between 3 and 5 nm to the thickness of the surface layer, consistent with the addition of a single leaflet of the cell membrane bilayer. Environmental scanning electron microscopy (ESEM) was used to confirm that the resulting surface was uniformly hydrophilic, indicating that cell membrane material completely covered the surface33 (Figure 8). The composition of the HBMs formed from RBC ghosts was investigated by examining with SPR the effect of enzymes on the apparent thickness of the bilayer.33 Treatment with neuriminidase, an enzyme that cleaves sialic acid residues, resulted in a 0.2 nm decrease in the thickness of the bilayer. This is consistent with an estimate of 8 mol % of dry weight of the red blood cell membrane that is attributed to sialic acid residues. The addition of chymotrypsin resulted in an initial increase in the apparent thickness of the HBM, which was followed by a loss of material from the surface.
(38) Dempsey, C. E. Biochim. Biophys. Acta 1990, 1031, 143-161. (39) Tosteston and Tosteson. Biophys. J. 1981, 36, 109-116.
(40) Petralli-Mallow, T.; Plant, A. P.; Lewis, M. L.; Hicks, J. M. Submitted.
Figure 7. Surface-enhanced resonance Raman spectroscopy (SERRS) of cytochrome c bound to negatively charged HBMs containing 20% deuterated dioleoylphosphatidic acid/80% deuterated dimyristoylphosphatidylcholine over a hexanethiol monolayer. As increasingly negative potentials were applied, Fe in the cytochrome heme was reduced as indicated. Bands at 1584 and 1644 cm-1 indicate that the low-spin conformation of the heme moiety,42 which is the native solution structure, is retained on HBMs. At a bare Ag electrode, cytochrome c assumes the high-spin state as it is reduced, as indicated by the peak at 1630. Potentials are versus Ag/AgCl.
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Figure 8. Red blood cell membrane hybrid bilayers. Alkanethiol-coated gold surfaces were floated on buffer containing RBC ghosts for >1 h and then rinsed and dried. (A) Atomic force microscopy indicates that a layer of material can be pushed by the tip by applying approximately 30 nN. (B) Environmental scanning electron microscopy of an alkanthiol-coated sample (right) and one to which RBC ghost membrane has been added (left). Increasing the pressure in the sample chamber causes water to condense onto the cooled samples. The membrane-treated sample appears uniformly hydrophilic over a very large area, while the monolayer sample on the right is hydrophobic.
Membrane protein activity is associated with this cell membrane hybrid. Acetylcholinesterase activity was measured colorimetrically. Cyclic voltammetry indicated that more redox current could pass through RBC membrane/decanethiol bilayers than through the decanethiol monolayer33 (Figure 9). This current could be blocked by the addition of 4,4′-diisothiocyanate stilbene-2,2′-disulfonic acid (DIDS), a specific blocker of Band 3, a channel protein common in RBC membranes.
Future of Hybrid Bilayer Membranes The demonstration of the formation of RBC membrane hybrids is supported by data that indicate that formation of HBMs occurs similarly via vesicles or cell membrane particles and by analytical confirmation of the structure and composition of the RBC cell membrane hybrid. This demonstration has significant implications because it suggests that rugged membrane mimics can be selfassembled from complex biological membranes and that
Supported Hybrid Bilayer Membranes
Figure 9. Reduction of ferricyanide as a measure of the insulating properties of the surface layer. Inset: compared to a bare electrode, the decanethiol monolayer blocks electron transfer, resulting in less current which appears at a larger applied potential. The effect is greater when a layer of phospholipid is added to form an HBM. Adding a layer of RBC membrane does not have the same effect. After the addition of the RBC membrane layer, the current increases and is observed at a potential closer to the formal potential for ferricyanide (s) compared to the current that passes through a decanethiol monolayer (s). When DIDS (a specific blocker of Band 3 channel protein) is added, current is reduced (‚‚‚).
they will accurately reflect the composition of the starting material. This knowledge opens up the possibility of constructing rugged model membranes with a desired function without having to purify the functional molecule or even identify it. This approach will provide a new way of addressing questions concerning activity of cell membrane proteins and will be particularly relevant to measuring kinetics and binding constants for orphan receptor-ligand interactions. We have demonstrated that HBMs are compatible with commercial surface plasmon resonance instrumentation for this purpose.27 The further development of HBMs for functional studies, structural studies, and applications such as sensors requires optimizing the tethering chemistry to maximize
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ruggedness, while at the same time presenting a lipid matrix that is in a fluid, liquid-crystalline phase. For electrochemical detection, a matrix with a high electrical resistance is desirable. For inserting transmembrane proteins, providing a sufficiently large aqueous compartment on both sides of the tethered bilayer is highly important. Discovering chemistries that provide the desired characteristics has been the subject of an expanding body of literature. A large focus has been on different chemistries for “spacer” moieties between the gold support and the lipid bilayer. Efforts include spacers based on trioxaundecandioic acid and cysteamine,29 a systematic series of trioxaundecanol and succinic acid residues,22 peptides,18,24 an amphiphilic copolymer,5 and ethylene oxide-linked phospholipids,23 alkanes,41 and cholesterol.21 Biomimetic lipid bilayers based on thiol tethering to a metal surface have been accepted as biologically relevant model membranes. There has been a substantial demonstration of success in constructing complex tethered lipid bilayer systems that include functional membrane proteins, examples of which have been mentioned here. A commercial electronic sensor based on this approach is the subject of a vigorous developmental effort.20 A significant contribution to the popularity of thiol-tethered bilayers is the wide array of analytical options that the approach presents. Ongoing work in this area will lead to improvements of matrix characteristics, new approaches to the architecture of the solid supports, and the further development of quantitative and structural analytical techniques. Validation and improvements in lipid matrix characteristics will lead to meaningful membrane protein structure/function studies. Variations on the constructs that have been presented to date will be important tools in future advancements in cell biology, protein structure/ function relations, pharmaceutical screening, and sensors. Acknowledgment. The author is greatly appreciative of the many collaborators who have contributed to work from this laboratory. Any mention of specific products is for clarification only and does not constitute an endorsement by NIST. LA981662T (41) Vanderah, D. J.; Meuse, C. W.; Silin, V.; Plant, A. L. Langmuir 1998, 14, 6916-6923. (42) Hildebrandt, P.; Stockburger, M. J. Chem. Phys. 1986, 90, 60176024.
