Planar Bilayer Measurements of Alamethicin and Gramicidin

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Planar Bilayer Measurements of Alamethicin and Gramicidin Reconstituted in Synthetic Block Copolymers Michael Martin, Timothy Dubbs, and Joel R. Fried Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03309 • Publication Date (Web): 01 Jan 2017 Downloaded from http://pubs.acs.org on January 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Planar Bilayer Measurements of Alamethicin and Gramicidin Reconstituted in Synthetic Block Copolymers Michael Martin, Timothy Dubbs and J.R. Fried

Abstract: This work explores methods for forming and characterizing biomimetic planar membranes composed of amphiphilic block copolymers. The membranes are diblocks and triblocks with hydrophilic blocks of poly(2-methyl-2-oxazoline) (PMOXA) and hydrophobic blocks of poly(dimethylsiloxane) (PDMS). Experiments with the lipid diphytanoyl phosphocholine (DPhPC) serves as a basis for comparison with the polymeric membranes. Phase-contrast microscopy is used to study how membranes evolve over time after their formation. Capacitance measurements as a function of thinned membrane area (prepared from two separate solvent systems) are performed to clarify the importance of the Plateau-Gibbs border on electrical measurements. Finally, functional reconstitution of the two ion channels, alamethicin and gramicidin, is investigated. Imaging in transmitted phase-contrast mode provides visualization of thinned regions that contain monolayers or bilayers (in the case of diblock copolymer). The specific capacitance measurements yield 0.28 µF/cm2 with a corresponding thickness of 8.5 nm for PMOXA6 PDMS35-PMOXA6 (blocks of 6 PMOXA and 35 PDMS repeat units) formed from a solution of ethanol-decane, 0.55 µF/cm2 and 4.4 nm in chloroform-toluene and 0.46 µF/cm2 and 5.4 nm for the diblock PMOXA6 -PDMS17 in ethanol-decane. Alamethicin reconstitution in the block copolymers show slower channel forming kinetics with somewhat higher conductance values than found in DPhPC. Gramicidin in the block co-polymer shows a slightly voltage dependent conductance as a function of time, with little stochastic conductance state switching, in contrast to reconstitution in DPhPC where gramicidin switches states at ~3 Hz.

Introduction Exploiting the exquisite nanoscale engineering principles of biological systems outside of living organisms has the potential to revolutionize technologies such as energy generation, sensors, environmental remediation, drug delivery, nanoscale synthesis, robotics, material science, and even artificial intelligence. Using biologically derived components, e.g. lipids and proteins, researchers have already demonstrated that nanobiological systems can produce glucose from artificial photosynthesis (1), solar-powered antibiotic remediation (2), glucose-consuming biofuel cells (3, 4), and a variety of detection systems (5-7), to name a few. Invariably, such systems function for only a brief period of time, i.e. typically less than 24 hours. The biological molecules and the macromolecular assemblies in such devices are prone to mechanical instability and degradation by mechanisms that include oxidation and hydrolysis. These shortcoming are managed in organisms by the constant metabolic turnover of such molecules; a process that is

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

engineered to remove and replace labile molecules and one that is (so far) difficult to mimic in artificial biological constructs. In the case of systems that utilize lipid membranes as integral elements, one approach to overcoming the fragility of these 5-nm thick components has been to utilize carefully engineered block copolymers in their place. Studies of synthetic polymer membranes as a mimic for natural planar lipid bilayers began with some of the first single ion channel measurements of gramicidin by Hladky and Haden in 1972 (8). The field of biomimetic membranes has since grown to include short-chain amphiphilic block copolymers that can be engineered to self-organize into lamellar sheets, vesicles, or micelles, just as lipids, with the advantage of readily tuned thickness and enhanced chemicalmechanical stability. Additionally, crosslinking moieties can be synthesized within the amphiphilic molecules to allow resulting membranes to be crosslinked after the inclusion of proteins resulting in far greater membrane lifetime (9, 10). To date at least 8 different membrane proteins have been functionally reconstituted within block copolymer membranes, including aquaporins (11), α-hemolysin (aHL)(12), outer membrane protein G (OmpG)(9, 12) , outer membrane protein F (ompF) (13, 14), alamethicin (12), gramicidin (15, 16), maltoporin (9), bacteriorhodopsin (10, 17), and cytochrome C (17). Application of self-organized polymer structures with enzymes, proteins, and other biologically active molecules incorporated have been demonstrated in drug delivery (18-20), energy harvesting (21), remediation (21), nanoscale synthesis (22), and highly selective separations (11, 23). Block and especially triblock copolymers of poly(2-methyl-2-oxazoline) (PMOXA) and poly(dimethyl siloxane) (PDMS) are currently one of the most extensively studied polymer systems for nanobiological applications with PMOXA being hydrophilic and PDMS being hydrophobic (21, 24-26). Both AB and ABA block copolymers (where A is the hydrophilic and B the hydrophobic blocks) have been studied in polymersomes and to a lesser extent planar membrane conformations. PMOXA is a chemically stable amorphous polymer that remains soluble in aqueous solution from 0-100 °C with no lower cloud point. The PDMS segment offers a mixture of properties that includes chemical inertness, low permeability to macromolecules, and flexibility. Indeed, the ability of PDMS to adapt a compressed conformation has reportedly enabled reconstitution of proteins in membranes that are much thicker than the hydrophobic thickness of the protein (9, 11, 15, 27). Theoretical considerations indicate that due to the energetic penalty required to compress polymer membranes that are thicker than the lipid membranes, it is expected that the equilibrium concentration of adsorbing molecules will be lower than in natural membranes (28). Due to its small size, gramicidin reconstitution in synthetic membranes acts as a particularly useful metric for probing the limits and effects of thickness disparity between ion channels and block copolymers. Only two experimental studies have investigated gramicidin-mediated ion conduction through polyoxazoline and polydimethylsiloxane block copolymers. In one case, Lomora et al. (15) incorporated the channels into vesicles and the resulting conductance was measured using pH and ion sensitive fluorescent chemical probes on the interior. This study demonstrated that


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the gramicidin channel would not function in triblock copolymers that were thicker than 12.1 Å (PMOXA7-PDMS49-PMOXA7) but did not give an indication of how the kinetics of conduction (unit conductance and open probability) changed as the membranes became thicker nor was selectivity to monovalent ions demonstrated. A second study by Gonzalez-Perez et al.(16) focused on stabilizing a membrane of PMOXA7-PDMS60-PMOXA7 over a microfabricated array of holes, the authors claim to show single channel conductance of hydrogen ions through reconstituted gramicidin channels. They showed only a single trace with 4 very noisy unit conductance steps – the band width of the measurement was limited by the large capacitance of the microfabricated support, and no statistics on open probability were provided. It is interesting to note, as well, that the polymer used in the Gonzalez-Perez report was thicker than the upper limit established by Lomora et al. (15). Further, Gonzalez-Perez admit that they cannot rule out aggregation as the conductance mechanism. Aggregation of the ion channel might introduce detergent-like holes that would result in a lack of selectivity for monovalent ions. Some of the conflicting results between studies by Lomora et al and Gonzalez-Perez et al. might be explained if the triblock molecules are taking on different conformations when they are in planar membranes and polymersomes. Specifically, it is known that lamellar phases of triblock polymers may self-organize into sheets with either a bridge conformation, where the hydrophobic moiety of individual molecules spans the hydrophobic region, or in a loop configuration, where the hydrophobic block turns on itself so that the hydrophilic ends of a polymer molecule are on the same side of the sheet (29-31). There is some experimental indication that the loops may become interdigitated to form membranes less than the bridge configuration thickness (21, 32). Coarse-grained molecular dynamics simulations were performed by Srinivas et al. (33) using triblocks of poly(ethyl ethylene) and poly(ethylene oxide) (PEO-4-PEE-16-PEO-4) to explore the effects of chain flexibility on membrane ion channel behavior. They found that the majority of molecules formed in cylindrical polymer structures were of the loop conformation, though they were not interdigitated, thus the membrane was at least as thick as the length of bridge conformation structures. Choi et al. (32) investigated thickness differences between measurements of polymersomes using cryoTEM and planar membranes using impedance spectroscopy. The triblock used in this study was PEOXA11PDMS75-PEOXA11, where PEOXA is poly(2-ethyl-2-oxazoline) and MW: 1100-b-5600-b-1100, Mw/Mn = 1.48. For cryoTEM imaging, polymer vesicles were formed in a mixture of DI water and ethanol then dried in a desiccator before imaging. The authors conclude that the PDMS block length was 3.8±0.4 nm. Using the planar bilayer technique with the polymer solvated in chloroform-toluene the authors measure a thickness of 9.3-8.8 nm. The study concludes that these differences in thickness may be due to the copolymer taking an intercalated loop midblock structure where the loops are interdigitated as opposed to forming a bilayer. This work examines methods for forming and characterizing PMOXA-PDMS membranes using standard bilayer lipid membrane (BLM) electrophysiology techniques. Diblock and triblock systems are explored along with a lipid that acts as a control for the experiments. White-


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

light microscopy is used to elucidate how membranes evolve over time after their formation. Capacitance measurements as a function of thinned membrane area (prepared from two separate solvent systems) are presented to clarify the equilibrium thickness and the importance of the Plateau-Gibbs border at the periphery of the membrane on electrical measurements. Finally, functional reconstitution of the two ion channels, alamethicin and gramicidin, is explored. Alamethicin is intended as a control experiment of our technique while experiments with gramicidin are intended to extend the work of Lamora et al.(15) and Gonzalez-Perez et al.(16).

Experimental Section ABA and AB block copolymers of poly(2-methyl-2-oxazoline) (PMOXA), and poly(dimethyl siloxane) (PDMS) were acquired from Polymer Source, Inc. The diblock was structured with block lengths of 6 PMOXA units and 17 PDMS units (PMOXA6 -PDMS17), MW:1300-b-500 and a polydispersity of 1.2 using a benzyl linker, part number P10649-DMSMEOXZ. The triblock composition was PMOXA6-PDMS35-PMOXA6 of MW: 550-b-2600-b-550 with a propyl linker and a polydispersity of 1.3, part number P14521D-MOXZDMSMOXZ. Reagents were chloroform (J.T. Baker), toluene (BDH), decane (99%+ pure from Alfa Aesar), ethanol (200 proof from Sigma-Aldrich), sodium chloride (Amresco), Tris HCl (G Biosciences), KCl (BDH), HEPES (99% pure, Acros Organics), and anhydrous DMSO (99.7%, Acros Organics). Gramicidin A (gA) and alamethicin (product number BML-A150) were from Enzo Life Sciences. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was purchased solvated in chloroform from Avanti Polar Lipids. Planar BLM experiments were performed using a commercially available 25-µm thick Teflon membrane with a single 0.08-0.12 µm diameter hole or a single 0.25-0.35 µm diameter hole (part number TP-01 and TP-03 respectively from Eastern Scientific, LLC. These were mounted in a horizontal bilayer chamber incorporating a glass window to facilitate membrane imaging via inverted microscopy, with the upper half composed of Teflon (BCH-1, Eastern Scientific, L.L.C.). Electrodes were chlorodized silver wire; via dipping in Clorox™ bleach for 2–5 min and in some experiments agar salt bridges were used to avoid deleterious effects of Ag ions on membrane proteins. Salt bridge electrodes were prepared using 4% agar in a 3 M KCl solution (12). Experiments were performed in a commercially available Faraday cage (Warner Instruments) on a vibration isolation table. Data were acquired using an Axopatch 200B patch clamp amplifier set to a 5 kHz sample rate with a 1 kHz 8 pole low pass Bessel filter and a Digidata 1440A data acquisition system both from Molecular Devices. Capacitance measurements were made by application of a triangular wave with a 20 mV amplitude at 30 Hz while recording the resulting square wave current. One deduces the capacitance using the fact that I = dQ/dt = C dV/dt where I is current, Q is charge on the capacitor (membrane) with capacitance, C and applied voltage V (34). The resulting current versus time data were then histogramed and Gaussian functions were fit to the peaks. To establish error in the measurement


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the 1σ value (standard deviation) from the curve fit was used. Images and area measurements were acquired using a Leica DMiL inverted microscope using both phase contrast and bright field modes at 200X. The area of the thinned region within membranes was measured by fitting an ellipse to the smooth area at the center using calibrated imaging software (AmScope). The measurement error at this magnification was estimated to be ±15 µm. Calculations of specific capacitance and membrane thickness assume that the Plateau-Gibbs boarder (the anular region around the thinned membrane) is thick enough that this capacitance is insignificant in comparison to the thinned area. This is typically a reasonable approximation for lipids in alkanes (34, 35) though, as demonstrated in this work, such an approximation may not hold for synthetic membranes of PMOXA-PDMS. Polymers were solvated in chloroform:toluene mixtures in a 2:3 ratio to a concentration of 2025 mg/ml (12, 26, 36). In an effort to avoid denaturing proteins due to aggressive solvents and explore other approaches, the polymers were also solvated in a 1:8 ethanol:decane solution to a concentration of 20-25 mg/ml. We chose not to use the chloroform-decane solvent system as in Gonzalez-Perez et al. (16) as the authors reported that the polymers were not fully solvated. DPhPC was prepared by evaporating the chloroform in a vacuum oven at room temperature for 1 hour at -600 Torr then it was dissolved in decane and vortexed for an hour at a concentration of 20mg/ml. Alamethicin was solvated in ethanol at a concentration of 10 ng/ml. Gramicidin A was reconstituted using a solution of 1:1 DMSO:EtOH at a concentration of (240 µM) 0.45 mg/ml per Lomora et al (15) and for some experiments a 12n M (22.59 x 10-6 mg/ml) solution in ethanol was used (16). Buffers were 0.5 M KCl, or 1 M KCl, 5 mM HEPES at pH=7.3. Before forming membranes, the aperture was conditioned by applying 5-10 µl of solvated block copolymer or lipid solution and allowing it to dry for 30 minutes. Then each side of the chamber was filled with 0.75 ml of buffer and the membrane was formed over the supporting PTFE sheet using a flame polished micropipette to spread ~5 µl of lipid or polymer solution over the aperture. Protein was incorporated into the membrane either by direct addition to the solvated polymer solution or by adding it to the aqueous phase on either side of the membrane.

Results and discussion Membrane Thinning and Visualization When natural or synthetic amphiphiles are initially spread over an aperture from solutions of organic solvent, the suspended membrane contains a large amount of solvent. In the case of lipids, free energy minimization causes solvent to leave the membrane allowing the leaflets to join forming a bilayer (34). Some of the solvent collects at the interface between the membrane and support in the Plateau-Gibbs border (35, 37). To explore this process in polymers, the morphology and the thinned area of the membranes were monitored during experiments using the inverted microscope. Unlike DPhPC, in general the synthetic membranes did not readily thin to monolayers (or bilayers in the case of diblocks). Often the resulting membranes would


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

remain thick for hours until their eventual rupture. We found it useful to use an air bubble created at the tip of a flame polished glass capillary to induce thinning; presumably pushing excess material to the periphery of the hole. (See video S1) Once sufficient material was removed from the aperture, the membrane would thin of its own accord, driven by free energy minimization or an applied voltage. It should be noted that the membrane area clearly increased as a function of DC voltage, necessitating regular measurements of membrane dimensions. Figure 1 illustrates the thinning of PMOXA6 -PDMS35-PMOXA6 from an ethanol:decane mixture in 0.5M KCl at pH 7.3 for the TP-03 aperture. The final frame depicts the membrane just before rupture. The images were recorded using phase contrast in transmitted light. Both lipid and synthetic membranes formed in this work showed a similar morphology with an annular Plateau-Gibbs border that ranged in width from 10 – 100 µm.

Figure 1 An A6B35A6 triblock membrane thinning in buffer over a 335 µm diameter aperture in a 25 µm thick Teflon sheet. The photomicrograph was taken in transmitted light using phase contrast.

Specific Capacitance Measurements in Triblock and Diblock Polymers Careful measurements of capacitance were made to determine the specific capacitance (per unit area) and the thickness of the membranes. Stray capacitance of the electrodes and chamber were measured using the same volume of electrolyte used in reconstitution experiments and a Teflon membrane of the same thickness without a perforation. For experiments using only AgCl wires the stray capacitance was 4.34 ± 0.178 pF, and with agar bridge electrodes the stray capacitance was 25.63 ± 0.739 pF. As a check on our calculations the hydrophobic thickness of a DPhPC membrane was determined to be 3.0 nm with a specific capacitance of 0.656 ± 0.0074 µF/cm2 using 2.20 as the dielectric constant for acyl chains (38). This hydrophobic distance is in reasonable agreement with two lamellar x-ray diffraction studies that reported values of 3.6 nm (39) and 3.4 nm for the phosphate-phosphate distance (40). Given that the electrical measurement is sensitive to only the hydrophobic distance in the membrane, one would expect


Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

our value to be somewhat less than those reported for the phosphate to phosphate spacing. Our thickness measurement disagrees with the work of Wong et al (12) who reports 5.2 nm but used a dielectric constant of 3. The specific capacitance also agrees well with values measured by Venkatesan et. al. of 0.685 ± 0.068 µF/cm2 for DPhPC using the droplet interface bilayer technique (41), and the value reported by Peterman et. al. of 0.64-0.70 µF/cm2 (42) via planar bilayer measurements. It is noteworthy that Peterman et. al. did not image the membrane and made no effort to compensate for the Plateau-Gibbs border at the edge of the membrane. These membranes tended to rupture at 250-300 mV.

Figure 2 Specific capacitance versus apparent thinned membrane area for di- and triblock copolymers from an ethanol-decane solution in 0.5 M KCl, pH 7.4. Specific capacitance versus apparent membrane area is presented in Figure 2 for the diblock and triblock copolymers formed from a solution of ethanol-decane. Data were taken in the 0.5 M KCl buffer at pH 7.4 and 5 mM HEPES. Experiments for the triblock were performed with the TP-03 aperture and for the diblock the TP-01 was used. Measurements of specific membrane capacitance seemed to converge to a constant value as the apparent thinned membrane area occupied a larger fraction of the entire aperture; as might be expected if the annulus of the membrane was contributing a non-negligible capacitance to the measurement. In the case of the ethanol-decane triblock, the data converge to a specific capacitance of 0.28 µF/cm2 and a corresponding thickness of 8.5 nm. For the diblock the data converge to 0.46 µF/cm2 and 5.4 nm. Similar experiments were performed on triblocks in the chloroform-toluene system (data not shown) and indicated convergence to a specific capacitance of 0.55 µF/cm2 and a corresponding thickness of 4.4 nm. In the above calculations a dielectric constant of 2.7 (12) was used for the PDMS layer. Diblock membranes formed out of ethanol-decane mixtures tended to rupture at 800 mV, ethanol-decane triblock membranes at 500 mV, and triblocks in chloroform-toluene tended to rupture at 350 mV. It is noteworthy that block copolymer


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

membranes typically did not rupture until the diameter of the thinned region became an appreciable fraction of the supporting aperture. In all tests the resistance of the membranes was >1 GΩ and often greater than 100 GΩ. Alamethicin Reconstitution Alamethicin is an antibiotic polypeptide containing 20 amino acids with a primarily α-helical structure that is synthesized by the fungus Trichoderma viride (43). It is known to spontaneously insert into natural (44, 45) and synthetic membranes (12) from an aqueous solution to form a voltage-dependent ion channel. Alamethicin is a member of a unique group of biologically active peptides known as peptaibols that contain non-proteinogenic amino acids (i.e. not coded for genetically) such as α-aminoisobutyric acid in high proportions. Channels are thought to form according to a barrel-stave model where the protein monomers act as the staves of a barrel penetrating the membrane. As a result, the channels can adopt at least 7 separate conductance states depending on the number of monomers incorporated into the pore (44, 46). Before a channel forms however, the proteins are adsorbed onto the membrane surface where evidence indicates they are incorporated into the headgroup region of lipids. As monomers accumulate on the surface, the bilayer is expanded laterally resulting in a reduction of the membrane thickness and providing space for the insertion of the proteins’ hydrophobic moieties (47, 48). Further, the literature indicates a critical ratio for protein:lipid concentration above which the protein becomes incorporated into the membrane and below which it remains on the surface (40, 47, 49) though these studies do not investigate voltage dependent gating. Thus it is apparent that membrane interactions with adsorbed alamethicin monomers are closely tied to the kinetics of pore formation. Indeed, it has been demonstrated from some of the earliest studies that channel kinetics and accessible conductance states are a function of membrane composition (46). Alamethicin has been studied in synthetic block copolymers by several groups. Haefele et al. (50) have studied alamethicin incorporation into PMOXA16 –PDMS74-PMOXA16 and PMOXA13 –PDMS23-PMOXA13 via Langmuir-Blodgett techniques coupled to Brewster angle microscopy. They report that the longer PDMS blocks facilitated insertion of alamethicin due to its higher flexibility and also conclude that high surface pressures in triblocks can prevent collapse of the peptide into the film. Wong et al. (12) reported alamethicin reconstitution in a triblock using BLM electrophysiology experiments and comment that channels required much longer to incorporate into a polymer membrane than a lipid membrane. They further reported studies of two lengths (PMOXA13–PDMS33-PMOXA13, 5.7 nm and PMOXA12–PDMS54-PMOXA12, 9 nm) but do not clearly specify which triblock successfully reconstituted the alamethicin. To introduce alamethicin into the membranes, at least 30 µl of the ethanol/alamethicin solution was introduced on the Trans side of the membrane in 0.5 M KCl with 5 mM HEPES (pH 7.3), per Wong et al (12). Experiments were also performed with 1 M KCl but the protein seemed to more readily incorporate into membranes bathed in lower ionic strength electrolyte, particularly in the case of the polymer. Figure 3 illustrates a representative current versus time


Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

plot for an alamethicin insertion event in both DPhPC and triblock copolymer. The right side of the figure depicts histograms of the current through the membrane for the two cases. For DPhPC, 5 separate conductance states are shown and for the triblock at least 6 are visible. Some of these conductance states may be due to the presence of more than one conducting alamethicin aggregate. In both the polymer and DPhPC cases, the noise in the data seems to increase once a channel has opened. The short transient current spikes are likely due to stochastic changes in the number of alamethicin monomers occupying the channel. It is clear that the peak conductance values are somewhat higher and the channel switch rates are faster in DPhPC compared to the triblock. Wong et al. (12) reported conductance states in triblock co-polymer at 0.3,1.1, 2.2 and 3.34 nS which roughly agrees with the values reported by Romer et al. (51) of 0.234, 1.13, 2.45, 4.02, and 5.93 nS in DPhPC suspended on porous alumina. In this work, conductance states were found at 56, 203, 350, 414, 650, and 728 pS for the triblock copolymer. These values are, however, closer to those reported by Heitz et al (52) of 40, 93, 147 and 173 pS in DPhPC and to the seminal work on alamethicin single channel activity by Sakmann and Boheim (46) of 20, 110, 250, 430 and 620 pS in rat and frog muscle cell membranes. It is well known that variations in the apparent conductance values and kinetics are a function of protein concentration (40, 52), membrane composition (46), fluidity (52), and (in the case of block copolymers) a function of the hydrophobic block length (50).


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 20

Figure 3 Reconstitution of alamethicin ion channels in DPhPC (top) and triblock copolymer (bottom). Graphs on the left of the figure shows conductance events verses time and on the right, histograms of the time dependent data. Gramicidin Experiments in Triblock and Diblock Copolymers Gramicidin was one of the first ion channels measured with electrophysiological techniques to single channel resolution (8) and its structure has been solved to high resolution (see PDB entries: 1MAG and 1GRM) in a number of conformations. In many ways it is the prototypical ion selective channel (53, 54) conducting only monovalent cations. Due to its very small size (only 15 residues and 13 Å long) monomers in each leaflet of a lipid bilayer must associate into an end-to-end dimer before an ion channel is formed (53). The stochastic association of these dimers results in very well defined steps of constant conductance when a membrane laden with the channels is examined under constant potential in electrophysiology experiments. Gramicidin’s ruggedness and ease with which it can be introduced to membranes by simply adding it to the adjacent aqueous phase surrounding the membrane has contributed to


Page 11 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

its utility as a research tool. In this work, it serves as a useful metric for exploring the lower limits of channel length in synthetic membranes. Typical gramicidin behavior in a lipid bilayer is shown in Figure 4. Gramicidin A was introduced to the electrolyte on either side of a preformed membrane from a 12 nM solution of ethanol (16). In this case the electrolyte was pH 7.4 1 M KCl as it is known to produce high conductance steps with gramicidin. After a brief incubation period (~10 min) the membrane becomes conductive and shows stochastic, discrete steps with a single channel amplitude of 22.6 pS, in agreement with published values (8).

Figure 4 Typical behavior of gA in a lipid bilayer. Conductance steps are 22.6 pS. In contrast to lipid membranes, gA (from either ethanolic solutions or DMSO:ethanol) was not readily absorbed into the polymer membranes when introduced via the adjacent electrolyte. Instead, it was necessary to add the gA directly to the polymer solution in order to produce an effect. The graphs in Figure 5 were produced by adding 7 µl of gA solvated in DMSO-ethanol at 240 µM directly to 50 µl of the polymer solutions. Control experiments were performed by adding DMSO:EtOH to the membrane solutions; these showed no significant


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

effect on the membranes. Though not shown here, experiments were also performed by adding 5 µl of 12 nM gramicidin in ethanol to 50 µl of polymer solution; these yielded qualitatively similar results to the DMSO:EtOH case. Experimental parameters delivering gA in DMSOethanol were chosen to mimic conditions from Lomora et al. (15) and for the gA in ethanol, conditions were chosen to reproduce the work of Gonzalez-Perez et al. (16).


Page 12 of 20

Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5 Examples of gramicidin activity in triblok (top) and diblock membranes (bottom)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In many experiments, the conductivity of the membrane behaved as though it was voltage gated with a threshold voltage of 200 – 400 mV. In the top graph of Figure 5, the conductivity of the membrane was roughly constant until the voltage was raised to 350 mV where stochastic steps in the conductance were observed. In contrast to reconstitution in lipids, gramicidin in block copolymers rarely exhibits obvious switching behavior between open and closed states as in Figure 4. That is, once conductive states are formed they seem to remain in the conductive state with time constants much longer than found in lipid. Discrete changes in current are observed but are not of constant height and do not seem to be multiples of some minimum conductance. The smallest conductance steps observed in 1 M KCl was 16 pS for the diblock and 9.1 pS for triblock solvated in chloroform-toluene; compared to 22.6 pS for DPhPC in 1 M KCl.. Additionally, it was not unusual to find large discrete drops in conductance on the order of 0.1-1 nS at a given voltage, see for example the end of the trace in the bottom of Figure 5. Based on experience with gA in DPhPC such effects are likely due to aggregates of gA molecules leaving the thinned area of the membrane via lateral diffusion, most likely moving to the Plateau-Gibbs border at the outer edge of the membrane. Gramicidin conductance was never observed for triblock reconstituted from ethanol-decane; at 8.5 nm it was the thickest of the polymer membranes tested in this work. The graphs in Figure 5 represent a small fraction of the observed behavior in gramicidin loaded membranes over the course of these experiments. Often the membranes remained highly resistive (> 1 GΩ) or if they were conductive, the current at a given voltage was approximately constant as a function of time. Possible causes for this variety of observations may be linked to variation in membrane thinning, polymer molecules taking on a variety of conformations, poorly controlled lateral film stress, or the gramicidin being sequestered to regions outside the thinned membrane area.

Conclusions Planar membranes were formed with synthetic AB and ABA block copolymers of PMOXA and PDMS using BLM techniques for their stabilization. Transmission light microscopy was used to visualize their evolution over time and to monitor the morphology of thinned (presumably solvent free) area. It was demonstrated that in many cases polymer membranes did not thin of their own accord, in contrast to suspended membranes of lipid bilayers. Instead, the polymer suspended membranes would often remain thick, i.e. not forming a true bilayer or monolayer, until their sudden rupture. This finding is consistent with the lower lateral diffusion constants reported in block copolymers versus lipids; 2.35 µm2/s in PMOXA6PDMS34-PMOXA6 verses l2.5 µm2/s in POPC (55). A technique was developed to accelerate thinning by pushing on the membranes with an air bubble trapped at the end of a flame polished glass pipette. Capacitance, resistance and rupture voltages of polymer membranes were measured using BLM electrophysiology techniques while simultaneously imaging the membranes in an inverted


Page 14 of 20

Page 15 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

microscope to carefully measure the thinned area. These experiments showed that the specific capacitance was a monotonically decreasing function of apparent thinned membrane area that converged to an approximately constant value as the diameter of the thinned spot became a larger fraction of the total aperture diameter. These data indicate that in contrast to lipid membranes, the capacitance of the Plateau-Gibbs border contributes a non-negligible capacitance to the total system. Therefore it seems likely that this region is significantly thinner than in the lipid bilayer case. The measurements for the hydrophobic thickness of the three polymer/solvent systems from the capacitance values were: 8.5 nm (0.28 µF/cm2) for the ethanol-decane triblock, 5.4 nm (0.46 µF/cm2) for the ethanol-decane diblock, and 4.4 nm (0.55 µF/cm2) for the triblock in chloroform-toluene. The value reported for the ethanol-decane triblock matches well with the 9.2 nm measured in polymersomes of PMOXA6 –PDMS34-PMOXA6 using cryoTEM by Lomora et al. (15) and our value of 4.4 nm agrees well Wong et al. (12) who report 5.7 nm with PMOXA13 –PDMS33-PMOXA13 for membranes in a planar configuration. Although these two studies looked at different configurations (polymersome verses planar) it is noteworthy that Lomora et al. prepared the polymersomes from a solution of ethanol and that Wong et al. used a solution of chloroform and toluene. Measurements of rupture voltage showed that the diblock from ethanoldecane (5.4 nm thick) ruptured at 800 mV, triblock from ethanol-decane (8.5 nm) at 500 mV and triblock from chloroform-toluene (4.4 nm) at 350 mV. Such a counter intuitive finding may be related to higher stability at the periphery of the membrane (the Plateau-Gibbs border) for the diblock which can split in to separate leaflets to compensate for the difference between the membrane thickness and supporting Teflon sheet. The synthetic membranes did withstand higher voltages than lipid based membranes though their lifetimes (once thinned) were comparable to that of lipid membranes and is consistent with observations of Nardin et al. (26) who found that rupture dynamics of PMOXA-PDMS membranes were similar to lipid membranes (26). Also, they found a wide range of defect onset times in PMOXA-PDMS and comment that there maybe be local conformational disorder that is responsible. This finding is consistent with both the findings presented here and the hypothesis that these systems may be adapting loop or bridge conformations. Further, studies of surface pressure-area isotherms in LB experiments for polymers with a PDMS group as short as 23 repeats show that they are capable of adopting loop conformations at low surface pressure but tend to adopt bridge conformations at higher pressure (50). Reconstitution of two ion channels in the polymer membranes were investigated. Alamethicin was successfully introduced and showed gating properties qualitatively similar to those found in DPhPC lipid bilayers. Specifically, the conductance was approximately the same but the rate at which the channels changed state (added or lost a monomer) was nearly an order of magnitude longer in the polymer than in lipid. Gramicidin was also incorporated and found to have very different properties in polymeric membrane than in lipid bilayers; the conductance seemed significantly lower and there was virtually no evidence of conductance state switching.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Additionally, the experiments showed no gramicidin conductance for the thickest membrane at 8.5 nm. Further studies should focus on determining whether the gA conductance shows selectivity to monovalent ions in polymer as it does in lipids. Such experiments would rule out the possibility that conductance is occurring via aggregation of monomers to form surfactant-like pores. Differences in ion channels reconstituted in polymer and lipids likely stem from a number of differences between the two materials. The lack of gA incorporation via the electrolyte is consistent with reports that poly(2-methyl-2-oxazoline) prevents proteins from adsorption (56). Also, the polymer’s polydispersity may allow shorter chains to segregate from longer chains (57) that could enable or hamper membrane protein reconstitution mimicking the microdomains found in biological membranes (58, 59). A dependence of protein function on polydispersity might cause differences in reproducibility between research groups or from batchto-batch. Further, it is widely accepted that the gramicidin dimer, with a hydrophobic thickness of 22 Å (53) causes the typically longer fatty acid chains of lipids to deform in the region around the ion channel (53, 54) and it is evident based upon the work of Lomora et al. (15) that a similar mechanism occurs in the block copolymer system up to a maximum thickness. In the pioneering work of Hladky and Haydon (60), the conductance of the gramicidin channel was shown to be constant as a function of membrane thickness except for the thickest membrane in their study (at 6.3 nm) where they found a sub-conductance state that was roughly half the value of thinner membranes. Thus it is reasonable to expect that the conductance would be impacted by the thicker polymer membranes. The conductance of gA may be further reduced due to interference with ion flow at the mouth of the ion channel by chains of PMOXA as was demonstrated for block copolymers of ethyl ethylene and ethylene oxide via coarse graining simulations (33). Additionally, surface charges on biological membranes, e.g. zwitterions in phosphocholine, have been shown to have an impact on the conductance of gA (61). The polymers in this work have no such surface charge; a property that might also be implicated in differences in protein incorporation from aqueous electrolyte. The slower dynamics for state switching in the alamethicin and (in the case of gramicidin) nearly zero state switching are likely, in part, related to the lower diffusion constant in polymer. Further it has been shown based on Langmuir-Blodget film studies that PDMS-PMOXA films may crystalize under conditions where the film is compressed very slowly (36).

Acknowledgments Appreciation is expressed for financial support of this work from the J.B. Speed College of Engineering, University of Louisville. References: 1. Wendell D, Todd J, Montemagno C. Artificial photosynthesis in ranaspumin-2 based foam. Nano Lett. 2010;10(9):3231-6.


Page 16 of 20

Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2. Kapoor V, Wendell D. Engineering bacterial efflux pumps for solar-powered bioremediation of surface waters. Nano Lett. 2013;13(5):2189-93. 3. Mano N, Mao F, Heller A. Characteristics of a Miniature Compartment0less Glucose-O2 Biofuel Cell and Its Operation in a Living Plant. J Am Chem Soc. 2003;125:6588-94. 4. Mano N, Mao F, Heller A. A Miniature Biofuel Cell Operating in A Physiological Buffer. J Am Chem Soc. 2002;124:12962-3. 5. Srivastava RC, Sahney R, Upadhyay S, Grupta RL. Membrane permeability based cholesterol sensor- A new possibility. Journal of Membrane Science. 2000;164:45-9. 6. Nikolelis DP, Pantoulias S, Krull UJ, Zeng J. Electrochemical transduction of the interations of the sweeteners acesulfame-K, saccharin and cyclamate with bilayer lipid membranes (BLMs). Electrochimica Acta. 2001;46:1025-31. 7. Futaki S, Zhang Y, Kiwada T, Nakase I, Yagami T, Oiki S, et al. Gramicidin-based channel systems for the detection of protein-ligand interaction. Bioorganic & medicinal chemistry. 2004;12(6):1343-50. 8. HLadky SB, Haydon DA. Ion Transfer Across Lipid Membranes Gramicidin A pt1. Biochimica et biophysica acta. 1972;274:294-312. 9. Meier W, Nardin C, Winterhalter M. Reconstitution of Channel Proteins in (Polymerized) ABA Triblock Copolymer Membranes. Angewandte Chemie International Edition. 2000;39(24):4599-602. 10. Lee H, Ho D, Kuo K, Montemagno CD. Vectorial insertion of bacteriorhodopsin for directed orientation assays in various polymeric biomembranes. Polymer. 2006;47(9):2935-41. 11. Kumar M, Grzelakowski M, Zilles J, Clark M, Meier W. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(52):20719-24. 12. Wong D, Jeon T-J, Schmidt J. Single molecule measurements of channel proteins incorporated into biomimetic polymer membranes. Nanotechnology. 2006;17(15):3710-7. 13. Zhang X, Fu W, Palivan CG, Meier W. Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane. Scientific reports. 2013;3:2196. 14. Nardin C, Thoeni S, Widmer J, Winterhalter M, Meier W. Nanoreactors based on (polymerized) ABA-triblock copolymer vesicles. Chemical communications. 2000(15):1433-4. 15. Lomora M, Garni M, Itel F, Tanner P, Spulber M, Palivan CG. Polymersomes with engineered ion selective permeability as stimuli-responsive nanocompartments with preserved architecture. Biomaterials. 2015;53:406-14. 16. Gonzalez-Perez A, Stibius KB, Vissing T, Nielsen CH, Mouritsen OG. Biomimetic triblock copolymer membrane arrays: a stable template for functional membrane proteins. Langmuir : the ACS journal of surfaces and colloids. 2009;25(18):10447-50. 17. Ho D, Chu B, Lee H, Montemagno CD. Protein-driven energy transduction across polymeric biomembranes. Nanotechnology. 2004;15(8):1084-94. 18. Felice B, Prabhakaran MP, Rodriguez AP, Ramakrishna S. Drug delivery vehicles on a nanoengineering perspective. Mater Sci Eng C Mater Biol Appl. 2014;41:178-95. 19. Zhang K, Tang X, Zhang J, Lu W, Lin X, Zhang Y, et al. PEG-PLGA copolymers: their structure and structure-influenced drug delivery applications. J Control Release. 2014;183:77-86. 20. Haggag Y, Abdel-Wahab Y, Ojo O, Osman M, El-Gizawy S, El-Tanani M, et al. Preparation and in vivo evaluation of insulin-loaded biodegradable nanoparticles prepared from diblock copolymers of PLGA and PEG. Int J Pharm. 2016;499(1-2):236-46. 21. Choi H-J, Montemagno C. Recent Progress in Advanced Nanobiological Materials for Energy and Environmental Applications. Materials. 2013;6(12):5821-56. 22. Spulber M, Baumann P, Saxer SS, Pieles U, Meier W, Bruns N. Poly(N-vinylpyrrolidone)poly(dimethylsiloxane)-based polymersome nanoreactors for laccase-catalyzed biotransformations. Biomacromolecules. 2014;15(4):1469-75.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23. Shen Y-x, Saboe PO, Sines IT, Erbakan M, Kumar M. Biomimetic membranes: A review. Journal of Membrane Science. 2014;454:359-81. 24. Zhang X, Tanner P, Graff A, Palivan CG, Meier W. Mimicking the cell membrane with block copolymer membranes. Journal of Polymer Science Part A: Polymer Chemistry. 2012;50(12):2293-318. 25. Mecke A, Dittrich C, Meier W. Biomimetic membranes designed from amphiphilic block copolymers. Soft Matter. 2006;2(9):751. 26. Corinne Nardin MWaWM. Giant Free-Standing ABA Membrane Langmuir : the ACS journal of surfaces and colloids. 2000;16:7708-12. 27. Stoenescu R, Graff A, Meier W. Asymmetric ABC-triblock copolymer membranes induce a directed insertion of membrane proteins. Macromol Biosci. 2004;4(10):930-5. 28. Pata V, Dan N. The Effect of Chain Length on Protein Solubilization in Polymer-Based Vesicles (Polymersomes). Biophysical Journal. 2003;85:2111-8. 29. Karatasos K, Anastasiadis SH. On the Loops-to-Bridges Ratio in Ordered Triblock Copolymers: An Investigation by Dielectric Relaxation Spectroscopy and Computer Simulations. Macromolecules 2000;33:523-41. 30. Matsushita Y, Nomura M, Watanabe J, Mogi Y, Noda I. Alternating Lamellar Structure of Triblock Compolymers of the ABA Type Macromolecules. 1995;28:6007-13 31. Gehlsen MD, Almdal K, Bates FS. Order-Disorder Transition: Diblock versus Triblock Copolymers Macromolecules 1992;25:939-49. 32. Choi H-J, Brooks E, Montemagno CD. Synthesis and characterization of nanoscale biomimetic polymer vesicles and polymer membranes for bioelectronic applications. Nanotechnology. 2005;16(5):S143-S9. 33. Srinivas G, Discher DE, Klein ML. Key Roles for Chain Flexibility in Block Copolymer Membranes that Contain Pores or Make Tubes. Nano Letters. 2005;5:2343-9. 34. Hanke W, Schulue WR, Sattelle DB. Planar Lipid Bilayers: Methods and Applications (Biological Techniques Series): Academic Press; 1994. 35. White SH. Analysis of the Torus Surrounding Planar Lipid Bilayer Membranes Biophysical J. _1972 v12.pdf>. Biophysical Journal. 1972;12:432-45. 36. Ho D, Chu B, Schmidt JJ, Brooks EK, Montemagno CD. Hybrid Protein–Polymer Biomimetic Membranes. IEEE Transactions On Nanotechnology. 2004;3(2):256-63. 37. White SH. A Study of Lipid Bilayer Membrane Stability Using Precise Measurements of Specific Capacitance. Biophysical Journal. 1970 10. 38. White SH. Formation of "Solvent-Free" Black Lipid Bilayer Membranes from Glyceryl Monooleate Dispersed in Squalene Biophysical Journal. 1978;23:337-47. 39. Hung WC, Chen FY, Huang HW. Order-disorder transition in bilayers of diphytanoyl phospatidylcholine. Biochimica et biophysica acta. 2000;1467:198-206. 40. Huang HW, Wu Y. Lipid-alamethicin interactions influence alamethicin orientation Biophysics Journal 1991;60:1079-87. 41. Venkatesan GA, Lee J, Farimani AB, Heiranian M, Collier CP, Aluru NR, et al. Adsorption Kinetics Dictate Monolayer Self-Assembly for Both Lipid-In and Lipid-Out Approaches to Droplet Interface Bilayer Formation. Langmuir : the ACS journal of surfaces and colloids. 2015;31(47):12883-93. 42. Peterman MC, Ziebarth JM, Braha O, Bayley H, Fishman HA, Bloom DM. Ion Channels and Lipid Bilayer Membranes Under High Potentials Using Microfabricated Apertures Biomedical Microdevices 2002;4(3):231-6 43. Leitgeb B, Szekeres A, Manczinger L, Vagvolgyi C, Kredics L. The History of Alamethicin: A Reveiw of the Most Extensively Studied Peptaibol Chemistry and Biodiversity 2007;4:1027-51. 44. Mayer M, Kriebel JK, Tosteson MT, Whitesides GM. Microfabricated Teflon Membranes for LowNoise Recordings of Ion Channels in Planar Lipid BIlayers. Biophysical Journal 2003;85:2684-95.


Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

45. Bruno Le Pioufle HS, Kazuhito V. Tabata, Hiroyuki Noji, and Shoji Takeuchi. Lipid Bilayer Microarray for Parallel Recording of Transmembrane Ion Currents. Analytical chemistry. 2008;80:328-32. 46. Sakmann B, Boheim G. Alamethicin-induced single channel conductance fluctuations in biological membranes Nature. 1979;282. 47. He K, Ludtke SJ, Heller WT, Huang HW. Mechanism of Alamethicin Insertion into Lipid Bilayers. Biophysical Journal. 1996;71:2669-79. 48. Wu Y, He K, Ludtke SJ, Huang HW. X-ray Diffraction Study of Lipid Bilayer Membranes Interacting with Amphiphilic Helical Peptides: DPhPC with Alamethicin at Low Concentrations Biophysical Journal. 1995;68:2361-9. 49. Chen F-Y, Lee M-T, Huang HW. Sigmoidal Concentration Dependence of Antimicrobial Peptide Activities: A Case study on Alamethicin Biophysical Journal. 2002;82:908-14. 50. Haefele T, Kita-Tokarczyk K, Meier W. Phase Behavior of Mixed Langmuir Monolayers from Amphiphilic Block Copolymers and an Antimicrobial Peptide. Langmuir : the ACS journal of surfaces and colloids. 2006;22:1164-72. 51. Romer W, Steinem C. Ipedance Analysis and Single-Channel Recordings on Nano-black Lipid Membranes on Nano-Black Lipid Membranes Based on Porous Alumina Biophysical Journal. 2004;86:955-65. 52. Heitz BA, Jones IW, Hall HK, Jr., Aspinwall CA, Saavedra SS. Fractional polymerization of a suspended planar bilayer creates a fluid, highly stable membrane for ion channel recordings. J Am Chem Soc. 2010;132(20):7086-93. 53. Kelkar DA, Chattopadhyay A. The gramicidin ion channel: a model membrane protein. Biochimica et biophysica acta. 2007;1768(9):2011-25. 54. Anderson OS, Koeppe RE, Roux B. Gramicidin Channels. IEEE Trans on Nanobiosience. 2005;4(1):10-20. 55. Itel F, Chami M, Najer A, Lörcher S, Wu D, Dinu IA, et al. Molecular Organization and Dynamics in Polymersome Membranes: A Lateral Diffusion Study. Macromolecules. 2014;47(21):7588-96. 56. Konradi R, Pidhatika B, Muhlebach A, Textor M. Poly-2-methyl-2-oxazoline: A Peptide-like Polymer for Protein-Repellent Surfaces. Langmuir : the ACS journal of surfaces and colloids. 2008;24:613-6. 57. Discher DE, Eisenberg A. Polymer vesicles. Science. 2002;297(5583):967-73. 58. Crane JM, Tamm LK. Role of Cholesterol in the Formation and Nature of Lipid Rafts in Planar and Spherical Model Membranes. Biophyical Journal. 2004;86:2965-79. 59. Cheng ZJ, Singh RD, Marks DL, Pagano RE. Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids. Molecular membrane biology. 2006;23(1):101-10. 60. Hladky SB, Haydon DA. Ion Transfer Across Lipid Membranes Gramicidin A pt1 Biochimica et biophysica acta. 1972;274:294-312. 61. Wang Z, Fried JR. A hierarchical approach for predicting the transport properties of the gramicidin A channel. Soft Matter. 2007;3(8):1041.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC Graphic


Page 20 of 20

Planar Bilayer Measurements of Alamethicin and Gramicidin

Recommend Documents