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S-layer Ultrafiltration Membranes: A New Support for Stabilizing Functionalized Lipid Membranes Bernhard Schuster,*,† Dietmar Pum,† Margit Sa´ra,† Orit Braha,‡ Hagan Bayley,‡ and Uwe B. Sleytr† Center for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, Universita¨ t fu¨ r Bodenkultur Wien, A-1180 Vienna, Austria, and Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, Texas 77843-1114 Received June 23, 2000. In Final Form: September 28, 2000 We report on a simple method to generate bilayer membranes on a smooth layer of crystalline bacterial cell surface (S-layer) protein lattices, deposited on a microfiltration membrane (MFM), the so-called S-layer ultrafiltration membrane (SUM). Folded bilayers, MFM-, and SUM-supported lipid membranes showed a specific capacitance of 0.6-0.7 µF/cm2. The S-layer produced a stabilizing effect on the lipid membrane; folded and MFM-supported membranes ruptured at the application of a first and a second voltage ramp, respectively, whereas the SUM-supported membranes could withstand multiple voltage ramps. The lifetime of the lipid membranes increased significantly in the order MFM-supported membrane < folded membrane < SUM-supported membrane. By contrast with MFM-supported lipid membranes, successful reconstitution of staphylococcal R-hemolysin (RHL) was observed in both folded and SUM-supported membranes. The unitary conductance of an RHL pore was similar when reconstituted in folded and in SUM-supported lipid membranes. As an alternative to hybrid and tethered lipid membranes, SUM-supported lipid bilayers provide a biomimetic environment for transmembrane proteins.
Introduction A key component in the combination of membraneassociated molecular recognition mechanisms with inorganic solids (e.g., gold-covered surfaces, silicon wafers, semiconductors, or optoelectronic devices) is an ultrathin layer separating the lipid membrane and the inorganic surface. The demands on this layer are manifold because it should both stabilize the lipid membrane and maintain the thermodynamic and structural properties of free membranes, thus providing an environment for the reconstitution and immobilization of proteins under nondenaturing conditions.1-3 In this study, crystalline bacterial cell surface layers (S-layers) have been used as supramolecular supporting structures for stabilizing lipid membranes. S-layers, composed of single protein or glycoprotein species, represent the outermost cell envelope component in organisms of almost every taxonomic group of walled bacteria and archaea. Pores in S-layer lattices are of regular size and morphology, and functional groups are aligned in welldefined positions and orientations.4,5 When isolated S-layer subunits are reassembled in vitro on lipid films, the repetitive protein domains of the S-layer lattice interact with lipid headgroups and significantly modulate the characteristics of the lipid film (particularly its fluidity and local order on the nanometer scale) without penetrat* Corresponding author: Center for Ultrastructure Research and Ludwig-Boltzmann-Institute for Molecular Nanotechnology, Universita¨t fu¨r Bodenkultur Wien, Gregor Mendel Strasse 33, 1180 Vienna, Austria. E-mail:
[email protected]. † Universita ¨ t fu¨r Bodenkulter Wien. ‡ Texas A&M University System Health Science Center. (1) Sackmann, E. Science 1996, 271, 43. (2) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105. (3) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58. (4) Sa´ra, M.; Sleytr, U. B. J. Bacteriol. 2000, 182, 859. (5) Sleytr, U. B.; Messner, P.; Pum, D.; Sa´ra, M. Angew. Chem., Int. Ed. Engl. 1999, 38, 1034.
ing the hydrophobic part of the membrane.6,7 Thus, the terminology ‘semifluid membrane’ has been introduced to describe such composite S-layer/lipid membranes.8,9 In this study, lipid membranes were generated directly on S-layer ultrafiltration membranes (SUMs) which had been produced by depositing S-layer fragments of Bacillus sphaericus CCM 2120 as a continuous layer on microfiltration membranes (MFMs) followed by chemical crosslinking of the lattice. The S-layer lattice of this organism has p4 symmetry (lattice constant, 12.5 nm), is composed of identical protein subunits (Mr ) 127 kD), and possesses pores 4.5 nm in diameter.10 SUM-supported lipid membranes were compared with folded bilayers and lipid bilayers generated on plain MFMs in terms of conductance, capacitance, and breakdown voltage. Further, the comparative reconstitution of R-hemolysin pores (RHLs) was examined. RHL is an exotoxin secreted by Staphylococcus aureus,11 which selfassembles on lipid bilayers to form a heptameric pore.12 This proof of lipid bilayer formation allowed an investigation of the benefit of the S-layer lattice as a supporting structure providing a biomimetic environment. Material and Methods Production of SUMs. B. sphaericus CCM 2120 was obtained from the Czech Collection of Microorganisms (CCM) Brno, Czech Republic. Growth of the bacteria and cell wall preparations were performed as described previously.10 To produce SUMs, S-layer(6) Schuster, B.; Pum, D.; Braha, O.; Bayley, H.; Sleytr, U. B. Biochim. Biophys. Acta 1998, 1370, 280. (7) Weygand, M.; Wetzer, B.; Pum, D.; Sleytr, U. B.; Cuvillier, N.; Kjaer, K.; Howes, P. B.; Lo¨sche, M. Biophys. J. 1999, 76, 458. (8) Pum, D.; Sleytr, U. B. Thin Solid Films 1994, 244, 882. (9) Gyo¨rvary, E.; Wetzer, B.; Sleytr, U. B.; Sinner, A.; Offenha¨usser, A.; Knoll, W. Langmuir 1999, 15, 5, 1337. (10) Weigert, S.; Sa´ra, M. J. Membr. Sci. 1995, 106, 147. (11) Bhakdi, S.; Tranum-Jensen, J. Microbiol. Rev. 1991, 55, 733. (12) Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. Science 1996, 274, 1859.
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carrying cell wall fragments were deposited on polyamide (Nylon) MFMs with an average pore size of 0.4 µm (SM 25058, Sartorius AG, Goettingen, Germany) as described elsewhere.13,14 SUMs were stored in Milli-Q-water (Millipore, minimum resistance >18M Ωcm), containing 50 mM NaN3 (Merck, Darmstadt, Germany) at +4 °C and were used within 12 days. For long storage (up to 1 year) SUMs had been soaked in 87% glycerol containing 50 mM NaN3 at +4 °C. The integrity of the ultrafiltration layer that consisted of S-layer fragments of B. sphaericus CCM 2120 was checked by filtration of a ferritin solution as described previously.10 All used SUMs showed a closed defectless ultrafiltration layer. Formation of Phospholipid Bilayer. The phospholipids 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidic acid (DPPA, both Avanti Polar Lipids) were dissolved in n-hexane/absolute ethanol 4:1 (v/v; both Merck). This stock solution, with a DPhPC:DPPA ratio of 4:1 (w/w) was stored at -20 °C at a concentration of 3.5 mg lipid per mL. A folded lipid bilayer of the lipid mixture was formed on a 150-µm orifice in 25-µm-thick Teflon film (Goodfellow, England) separating two compartments (2.5 mL each) of a planar bilayer apparatus.6,15,16 The shape and diameter of the orifice were imaged by scanning electron microscopy after sputtering with a thin layer of gold (Sputter coater SCD 004, Balzers, Lichtenstein; Zeiss DSM 940, Germany). At least 30 min before the formation of the DPhPC/DPPA bilayer, the aperture was pretreated with a small drop of hexadecane/pentane 1:10 (v/v; Fluka, Buchs, Switzerland). Both compartments were filled to just below the aperture with electrolyte (0.1 M KCl in Milli-Q-water; pH ) 6.6) with a bulk conductivity of (1.295 ( 0.01) S/m as determined with a conductivity meter (CDM206, Radiometer, Copenhagen). About 5 µL of the lipid stock solution was spread on the aqueous surface of each compartment and the solvent was allowed to evaporate for at least 20 min. The cis compartment was virtually grounded by an Ag/AgCl-electrode and voltage signs are referred to it; current is defined as positive when cations flow into this compartment. The trans compartment was connected to a patchclamp amplifier (EPC 9, HEKA Elektronik, Lambrecht, Germany). Raising the level of the electrolyte within the compartments to above the aperture led to formation of a lipid bilayer which was checked by measuring the conductance and capacitance of the membrane. All experiments were performed at room temperature (22 ( 2 °C). After each experiment, the Teflon aperture was cleaned extensively with chloroform, methanol, and ethanol and finally rinsed with Milli-Q-water. Formation of the Lipid Membrane on MFMs and SUMs. The two compartments of the bilayer apparatus were separated with a polyethylene plastic sheet (Saran Wrap, DowBrands Inc., Canada). This septum was cut to more than twice the size of the contact area of the faces of the compartments and folded in half. An orifice was made through the double layer of the plastic by punching with a perforating tool (0.4 mm in diameter).17 The orifices showed an area of (8.6 ( 1.2) × 10-4 cm2 as determined by light microscopy (100-fold magnification). A piece of MFM or SUM (15 × 15 mm) was placed over the aperture and between the two plastic layers (Figure 1). The side of the ultrafiltration membrane with the attached S-layer lattices pointed toward the cis compartment. Both compartments were filled to just above the aperture with electrolyte. The lipid stock solution was diluted 1:100 (v/v) with n-hexane/absolute ethanol (4:1, v/v). Subsequently, 5-10 µL of the diluted lipid solution were put on the aqueous surface of the cis compartment near the septum and the solvent was allowed to evaporate for at least 20 min. After lowering and raising the electrolyte level, lipid membrane formation was confirmed by measuring the conductance and capacitance. All experiments were performed at room temperature (22 ( 2 °C). Isolation and Purification of HL. Isolation and purification of HL was performed as described previously.18 In reconstitution
Characterization of the Folded and Supported Lipid Membranes. The conductance, capacitance, and breakdown voltage of SUM-supported membranes, (Figure 1) were measured and compared with those of folded and MFM-supported membranes (Table 1). All membranes were tight as the specific conductance was 500 2. ramp n.a. 210 ( 80 > 500 3. ramp n.a. n.a. > 500 (n ) 7) (n ) 4) (n ) 5) a Only one aperture was used throughout the whole set of experiments. n ) number of experiments.
of classical lipid bilayers (0.01-1 µS/cm2)21,22 or tethered membranes (0.5-7 µS/cm2).23 The specific capacitance of the folded membrane was calculated to be (0.7 ( 0.1) µF/cm2 which is in good agreement with data in the literature.22,24 MFM- and SUMsupported lipid membranes showed a value of (0.6 ( 0.1) µF/cm2 (Table 1). The specific capacitance of lipid membranes did not differ significantly in all three configurations and indicated bilayer formation because multiple lipid layers should give much lower values corresponding to an increased film thickness. It cannot, however, be completely excluded that the lipid membranes on the SUM, and especially on the much more corrugated MFM support, might be composed of a great number of bilayer regions bordering on thicker patches as it was reported for membranes on metal electrodes.25,26 But the formation procedure of the supported lipid membranes, where the porous support passed the lipid film floating on the air/ electrolyte interface only twice and the measured specific capacitance strongly indicate that the MFM- and SUMsupported lipid membranes consist of two lipid layers. A similar specific capacitance of 0.57 µF/cm2 has been reported for phospholipid monolayer deposited from the air/water interface onto an octadecanethiol monolayer.27 A phospholipid monolayer assembled onto an octadecanethiol monolayer by vesicle adsorption28 and a supported bilayer on a SnO2-electrode29 obtained specific capacitance values of (0.64-0.68) µF/cm2 and 0.6 µF/cm2, respectively. Tethered bilayer membranes on a solid support, generated from DPhPC derivatives,23 gave values of (0.50-0.68) µF/ cm2. The dielectric breakdown voltage of the folded lipid bilayer was determined to be (220 ( 40) mV by a first voltage ramp applied between 30 and 45 min after membrane formation (Table 1). This value is close to the reported breakdown voltage for other folded lipid bilayers,21 but lower than ∼370 mV for membranes generated (22) Hanke, W.; Schlue, W. R. In Planar Lipid Bilayers; Sattelle, D. B., Ed.; Academic Press: London, 1993; p 24. (23) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 4, 648. (24) Alvarez, O. In Ion Channel Reconstitution; Miller, C., Ed.; Plenum Press: New York, 1986; p 115. (25) Hianik, T.; Passechnik, V. I.; Sargent, D. F.; Dlugopolsky, J.; Sokolikova, L. Bioelectrochem. Bioenerg. 1995, 37, 61. (26) Stelzle, M.; Weissmu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974. (27) Steinem, C.; Janshoff, A.; Ulrich, W.; Sieber, M.; Galla, H. Biochim. Biophys. Acta 1996, 1279, 169. (28) Plant, A. L. Langmuir 1993, 9, 2764. (29) Zviman, M.; Tien, T. H. Biosens. Bioelectron. 1991, 6, 37.
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from a DPhPC/hexadecylamine lipid mixture19 and soybean lipid with cholesterol.20 MFM- and SUM-supported membranes revealed a higher stability than folded membranes because no irreversible rupture occurred during a first application of a voltage ramp. One may argue that there is a voltage drop across the porous support, and thus, the folded membrane may suffer a relatively high stress compared with the lipid membrane attached on the MFM and SUM. The main contribution to a resistance may come from the intact, continuous ultrafiltration layer, which is 20-50 nm thick and composed of up to three stacked S-layer cell wall fragments.30 The resistance of an SUM with an area of 1 cm2 was estimated from the lattice parameters and the mean pore diameter assuming that the pores within the SUM are filled with electrolyte and the MFM was considered to have no significant influence on the resistance. Because the estimated resistance of the SUM was about 5 mΩ, which is so low that it can be ignored, no significant influence of the adjacent SUM on the breakdown voltage is expected. Without applying voltage ramps, the folded membrane had a lifetime of up to 6 h as determined by measuring the conductance of the membrane every 20 min. The lipid membrane generated on the MFM could withstand a first voltage ramp up to 500 mV, but a second ramp applied approximately 1 h later caused irreversible rupture of the lipid membrane at a breakdown voltage of (210 ( 80) mV (Table 1). One exception was observed because this membrane could withstand a second voltage ramp. However, this membrane ruptured spontaneously only some minutes after the second voltage ramp. The lifetimes of the lipid membranes generated on the MFM were about 3 h. The SUM-supported lipid membranes did not rupture upon application of three voltage ramps of up to 500 mV (Table 1) and had lifetimes of up to 8 h. The difference in stability (breakdown voltage, lifetime) indicate distinct interactions of the lipid membrane with the MFM and the surface of the S-layer lattice of the SUM. The difference between the MFM and the SUM as supporting structures is the additional layer of 1-4 µm sized S-layer fragments deposited on the microfiltration membrane like a shingle roof.13 This makes the porous support smoother because the height of a single fragment is about 20 nm.30 The S-layer lattice must be a biomimetic and water-containing structure as the structural features of the bilayer lipid membranes are maintained. Assembly of rHL in Folded, MFM-, and SUMSupported Lipid Membranes. Functional heptameric RHL pores penetrate the nonpolar portion of a lipid membrane with a β barrel domain,31 which is ∼2.8 nm in height.12 Thus, RHL is able to span the hydrophobic domain of common phospholipid bilayers32 but not of multilayered structures. Therefore, bilayer formation can be proven by the presence of functional RHL pores. Successful reconstitution of RHL pores could not be obtained using MFM-supported lipid membranes, even after prolonged stirring periods, although this was expected from the specific capacitance that suggested bilayer structure. In contrast, folded and SUM-supported lipid membranes allowed reconstitution of functional pores (Figures 2, 3, and 4). Thus, the S-layer is not only a stabilizing structure for lipid bilayers, but also provides a biomimetic environment for domains of transmembrane (30) Pum, D.; Neubauer, A.; Sleytr, U. B.; Pentzien, S.; Reetz, S.; Kautek, W. Ber. Bunsen-Ges. Phys. Chem. 1997, 101, 1686. (31) Gouaux, E. J. Struct. Biol. 1998, 121, 110. (32) Benz, R.; Fro¨hlich, O.; La¨uger, P.; Montal, M. Biochim. Biophys. Acta 1975, 394, 323.
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Figure 2. Current steps of reconstituted RHL pores in a planar folded bilayer clamped at +40 mV. Each current step, except the first one where two pores are open, is caused by the opening of a new ionic pore into the membrane. The electrolyte solution consisted of 0.1 M KCl, pH 6.6, T ) 22 ( 2 °C.
Figure 4. Current-voltage characteristics of RHL pores in an SUM-supported lipid bilayer. The curve has been normalized to the mean current flowing through a single pore. Insert, Current response to the application of voltage pulses to an SUMsupported membrane containing 9 RHL pores. Voltage steps were from a zero holding potential to the final potential indicated in millivolts at each current trace. In line with Figure 3a, one pore closes at +100 mV. Other conditions are as in Figure 2.
Figure 3. Single-channel recordings of staphyloccocal RHL pores reconstituted (a) in an SUM-supported lipid membrane and (b) in a folded bilayer clamped at (100 mV. Two RHL pores are reconstituted in both membranes. At +100 mV, one RHL pore gets closed in the SUM-supported membrane (a). At -100 mV, one pore gets closed in the folded bilayer (b). Other conditions are as in Figure 2. Table 2. Intrinsic Features of rHL Pores Reconstituted in Folded and SUM-Supported Phospholipid Bilayers bilayer unitary pore conductance at +40 mV (G+) (pS) unitary pore conductance at -40 mV (pS) pore diameter; calculated from (G+) (nm) sign of the voltage where closure was observed
folded
SUM-supported
140.0 ( 2.9 (n ) 19) 112.5 ( 4.9 (n ) 19) 1.17 ( 0.03
127.5 ( 2.0 (n ) 17) 90.0 ( 2.2 (n ) 17) 1.12 ( 0.02
negativea (n ) 72)
positivea (n ) 38)
a In more than 97% of the recorded closures these events occurred at the sign of voltage given in this table. n ) number of experiments.
proteins protruding from the lipid membrane. Because functional RHL pores can be reconstituted in SUMsupported lipid membranes, a considerable part of the lipid membrane on the SUM must exist as a bilayer. The unitary conductance of an RHL pore was determined from current steps as pores assembled and inserted (Figure 2) or by the stepwise decreasing current caused by the closure of single pores (Figure 3). The specific conductance of a single RHL pore was determined at (40 mV (Table 2) because at higher voltages (>(80 mV) closing events have been observed. In line with data reported in other
studies,33,34 the unitary conductance of RHL pores reconstituted in folded and SUM-supported lipid membranes (Figure 4) was lower at a given negative voltage than at the same but positive voltage (Table 2). The unitary conductance of a pore and gating depends on many parameters, such as the bulk conductivity of the electrolyte, the presence of di- or trivalent cations, the pH value, the sign of the applied voltage, and the composition of the lipid membrane.33,34 Nevertheless, the diameter of the RHL pore was estimated from its conductance at +40 mV and was compared with data obtained under similar conditions.33 Assuming that the length of the RHL pore is 10 nm,12 the diameter of the pore was calculated to be 1.17 and 1.12 nm for the folded and SUM-supported bilayers, respectively (Table 2). Thus, the diameter of the pore, reconstituted in folded and SUM-supported bilayers was not only in accordance with the reported value of (1.14 ( 0.04) nm33 but also with the crystal structure analysis (narrowest diameter, 1.4 nm).12 The conductance of RHL pores is similar in folded and SUM-supported lipid membranes with an unitary conductance of ca. 130-140 pS at +40 mV and 90-110 pS at -40 mV in 0.1 M salt at a pH of 6.6. The generally slightly lower apparent conductance of RHL pores in SUMsupported bilayers compared with folded ones (Table 2) might be due to the added resistance of the porous SUM. However, this contribution has to be low in a series connection because the resistance of the SUM was estimated to be in the mΩ range. Also a change in the conformation of the pore would be improbable, because both bilayers are composed of the same lipids and bathed in the same electrolyte. The current flowing through RHL pores was measured at positive and negative potentials and, in accordance with other studies,33,34 a decreased conductance was found at a given negative potential compared with the same positive potential (Figure 4). Thus, the RHL pore is not a simple hole filled with aqueous solution, and it has been suggested that at least in a region of the pore the transport of ions is restricted.33 The voltage dependence of the unitary pore conductance indicated that the pores inserted in a uniform (33) Menestrina, G. J. Membr. Biol. 1986, 90, 177. (34) Korchev, Y. E.; Alder, G. M.; Bakhramov, A.; Bashford, C. L.; Joomun, B. S.; Sviderskaya, E. V. J. Membr. Biol. 1995, 143, 143.
S-Layer Stabilized Functional Lipid Membranes
orientation into the (symmetrical) bilayer. This behavior was found not only with folded but also with SUMsupported bilayers (Table 2). Closure of RHL pores, reconstituted in SUM-supported lipid membranes, occurred much more frequently (>97%, n ) 38) when a high positive potential (>+80 mV) was applied compared with the same negative potential (Figure 3a). In contrast, with folded lipid bilayers, closure of RHL pores occurred much more frequently (>97%, n ) 72) when a high negative potential (>-80 mV) was applied (Figure 3b). In accordance with another study on folded bilayer the closure of single pores was observed at a high positive potential.34 Reduction in RHL pore conductance at high transmembrane potentials (g100 mV) may be understood in terms of varying degrees of conformational rearrangement and closure at the stem base of the pore.12 The observed difference in the closing behavior of RHL pores reconstituted in folded and SUM-supported lipid bilayers might reflect the tightly attached S-layer lattice in the vicinity of the stem base. Site-directed mutagenesis and detailed structural and biophysical studies will be necessary to test and refine the molecular mechanisms for the pore closure at high transmembrane potentials. However, this issue was not the aim of this study. Conclusion This study demonstrated a simple method of assembly of bilayer membranes on two porous supports. The supported membranes are low conducting bilayers with a capacitance in the same range as folded bilayers, generated from the same lipids. In particular, the S-layer lattice on the SUM showed a stabilizing effect on the attached membrane compared with folded and MFM-
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supported lipid membranes as determined by the breakdown voltage and the lifetime. Most important, the reconstitution of RHL demonstrated bilayer formation on the SUM as the unitary conductance of the RHL pores was measured. Functionalization of SUM-supported lipid membranes with reconstituted transmembrane proteins such as ion channels with switches,18,35-37 engineered receptor proteins,2,38 or nanopores for nucleic acid sequencing39-42 might lead to practical applications in the field of biosensors. Furthermore, in the future, multiarrays of SUM-supported lipid membranes might be used at highthroughput screening.2,39,41 Acknowledgment. We thank Stefan Weigert from the Nanosearch Membrane GmbH for the preparation of the SUMs and for performing the filtration experiments, and Christopher Shustak and Stephen Cheley for the purified RHL. This work was supported by grants from the Austrian Science Foundation, Project S72/05 (U.B.S.) and Project 14419-MOB (D.P.); and by MURI (ONR) and TATP awards (H.B.). LA0008784 (35) Bayley, H. Curr. Opin. Biotechnol. 1999, 10, 94. (36) Cheley, S.; Braha, O.; Lu, X.; Conlan, S.; Bayley, H. Protein Sci. 1999, 8, 1257. (37) Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature 1999, 398, 686. (38) Stora, T.; Lakey, J. H.; Vogel, H. Angew. Chem., Int. Ed. Engl. 1999, 38, 389. (39) Deamer, D. W.; Akeson, M. Trends Biotechnol. 2000, 18, 147. (40) Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W. Biophys. J. 1999, 77, 3227. (41) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770. (42) Bezrukov, S. M. J. Membr. Biol. 2000, 174, 1.
