Incorporation of channel-forming peptides in a Hg-supported lipid bilayer

Journal of

Electroanalytical Chemistry Journal of Electroanalytical Chemistry 576 (2005) 121–128 www.elsevier.com/locate/jelechem

Incorporation of channel-forming peptides in a Hg-supported lipid bilayer Lucia Becucci a, Rolando Guidelli a,*, Cristina Peggion b, Claudio Toniolo b, Maria Rosa Moncelli a a

Department of Chemistry, University of Firenze, 50019 Sesto Fiorentino, Italy b Department of Chemistry, University of Padova, 35131 Padova, Italy

Received 23 July 2004; received in revised form 27 September 2004; accepted 30 September 2004 Available online 30 December 2004 Dedicated to Ron Fawcett on the occasion of his 65th birthday and in recognition of his contribution to Electrochemistry

Abstract The channel-forming peptides gramicidin and alamethicin were incorporated in a mercury-supported lipid bilayer composed of a tethered thiolipid monolayer with a self-assembled dioleoylphosphatidylcholine monolayer on top of it. The thiolipid consists of a hexapeptide chain with a high tendency to form a 310-helical structure, which terminates at the N-terminus end with a sulfydryl group for anchoring to the metal while the C-terminus end is covalently linked to the polar head of dimyristolylphosphatidylethanolamine. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiolpeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. Changes in the conductance of this biomimetic membrane following the incorporation of gramicidin and alamethicin were detected by impedance spectroscopy. The surface dipole potential of the hexapeptide chain and the transmembrane potential of the lipid bilayer were estimated by using a simple electrostatic model of the mercury|solution interphase.
2004 Elsevier B.V. All rights reserved. Keywords: Peptides; Electrochemical impedance spectroscopy; Lipid bilayers; Spacer; Thiolipids; Alamethicin; Gramicidin; Ion channels; Biomimetic membranes

1. Introduction For integral membrane proteins and channel-forming peptides incorporated in a metal-supported lipid bilayer to retain their functionally active state, the lipid bilayer should satisfy the following requirements: (i) it should be in the liquid crystalline state; (ii) it should have aqueous or hydrophilic media on both sides; (iii) it should be sufficiently free from pinholes and other defects that *

Corresponding author. Tel.: +39 55 457 3097; fax: +39 55 457 3098. E-mail address: guidelli@unifi.it (R. Guidelli). 0022-0728/$ - see front matter
2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2004.09.032

may provide preferential pathways for electron and ion transport across the bilayer [1]. The interposition of a hydrophilic layer between the metal surface and the lipid bilayer may serve to meet the second requirement and to allow a flow of ions across the metalsupported lipid bilayer incorporating channel-forming peptides or proteins. Both ‘‘hydrophilic spacers’’ and ‘‘thiolipids’’ have been frequently employed to create a hydrophilic environment on the metal side of the lipid bilayer. Hydrophilic spacers consist of polyethyleneoxy [2–6] or peptide chains [7] terminating at one end with a sulfydryl group for anchoring to a metal. Thiolipids consist

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HMDE and by self-assembling a dioleoylphosphatidylcholine (DOPC) monolayer on top of it was characterized by ac voltammetry with phase resolution and impedance spectroscopy. The suitability of this tethered film as a biomembrane model was tested by incorporating the channel-forming peptide antibiotics gramicidin and alamethicin. The role of the transmembrane potential on the function of these peptides was estimated on the basis of an electrostatic model of the mercury|water interphase [19,21].

of a hydrophilic chain anchored to the electrode surface at one end via a sulfydryl or a disulfide group, and covalently bound at the opposite end to the polar head of a phospholipid molecule [8–19]. Self-assembly of a lipid bilayer on top of a gold-supported hydrophilic spacer, or of a lipid monolayer on top of a goldsupported thiolipid, is usually achieved by spreading of lipid vesicles from an aqueous dispersion. On a mercury drop coated with a hydrophilic spacer [6,7] or a thiolipid [19], such a self-assembly is more easily carried out by immersing the drop in an aqueous electrolyte on the surface of which a lipid film had been previously spread. The present work describes the use of a novel thiolipid consisting of a hexapeptide chain with a high tendency to form a 310-helical structure [20], which terminates with a sulfydryl group for anchoring to the metal at the N-terminus end and is covalently linked to the polar head of a dimyristoylphosphatidylethanolamine molecule at the C-terminus end. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiolpeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. This thiolipid was anchored to a hanging mercury drop electrode (HMDE). Mercury shares with gold a strong affinity for sulfydryl groups, but it has the advantage of being readily renewable and of providing a defectfree surface to the self-assembling film. Moreover, its liquid nature allows the thiolipid molecules to move laterally with respect to each other, thus imparting to the metal-supported lipid bilayer a fluidity analogous to that of black lipid membranes. The biomimetic membrane obtained by anchoring this thiolipid to a

O

O

O

O O

O

CH2 HS

O

CH2 N H

Analytical and semi-preparative HPLC runs were performed on a Pharmacia model LKB-LCC 2252 liquid chromatograph equipped with an UVICORD model SD UV detector (226 nm) and a reverse-phase C4 Vydac model 214TP54 column (analytical) and model 214TP1010 column (semi-preparative). Mass spectra (MS) were obtained by electrospray ionization (ESI) on a Perseptive Biosystem Mariner API-TOF spectrometer. A 1 nM solution of neurotensin, angiotensin I and bradykinin in an acetonitrile + water 1:1 mixture, containing 1% formic acid, was used for calibration. The synthesis of the thiolipid HS-(CH2)2–CO-[AibGlu(OTeg)]2-Aib-Ala-DMPE (Aib, a-aminoisobutyric acid; DMPE, dimirystoyl-L -a-phosphatidylethanolamine; Teg, triethyleneglycol monomethyl ether), henceforth briefly referred to as TL (Fig. 1), was performed from Z-[Aib-Glu(OTeg)]2-Aib-Ala-OtBu (Z, benzyloxycarbonyl; OtBu, tert-butoxy), the synthesis of which was described elsewhere [7]. Acidolysis of this protected hexapeptide with a 1:1 s trifluoroacetic acid + methylene

O

O

H N

2.1. Synthesis of the thiolipid TL

O

O

O

2. Experimental

O

CH2 O H N

CH2 N H

O

H N

O

O

O H N

N H O

Fig. 1. Chemical structure of the thiolipid TL.

O O

O

O

P O CH2 CH CH2 OH

L. Becucci et al. / Journal of Electroanalytical Chemistry 576 (2005) 121–128

chloride solution afforded Z-[Aib-Glu(OTeg)]2-Aib-AlaOH. This peptide free acid was activated with HATU [O-(7-azabenzotriazol-lyl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (1 molar eq.) and N-ethyl,N,Ndiisopropylamine (DIEA) (2 molar eq.) in a 1:1 N,Ndimethylformamide (DMF) + tetrahydrofuran (THF) solution at 45 C. Reaction of this C-activated peptide with DMPE, dissolved in a 1:1 DMF + THF solution containing LiCl (20 molar eq.) at 45 C for 8 h, gave Z-[Aib-Glu(OTeg)]2-Aib-Ala-DMPE. The thiol, moiety was introduced into the peptide molecule by reaction of S-Trt(trityl)-protected 3-mercaptopropionic acid with the Na-deprotected peptide, the latter obtained by catalytic hydrogenation of Z-[Aib-Glu(OTeg)]2-Aib-AlaDMPE in methanol, in the presence of HATU (1 molar eq.) and DIEA (2 molar eq.) in methylene chloride. In the final step, removal of the Trt group was achieved by reacting the protected peptide with a 95:2.5:2.5 solution of trifluoroacetic acid + water + triisopropylsilane. The synthetic intermediates were purified by flash-chro˚ ; eluant methylene matography (ICN silica 32–63, 60 A chloride + methanol + acetic acid (6:1:0.5), while the final product was purified by semi-preparative HPLC. All products were characterized by MS: Z-[Aib-Glu(OTeg)]2-Aib-Ala-DMPE. ES-MS: m/ zcalcd: 1648.18 [M + H+], m/zfound: 1648.14 [M + H+]; m/zcalcd: 824.59 [M/2 + H+], m/zfound: 824.51 [M/ 2 + H+]. Trt-S-(CH2)2-CO-[Aib-Glu(OTeg)]2-Aib-Ala-DMPE. ES-MS: m/zcalcd: 1844.33 [M + H+], m/zfound: 1844.20 [M + H+]; m/zcalcd: 1865.33 [M + Na+], m/zfound: 1866.10 [M + Na+]. HS-(CH2)2-CO-[Aib-Glu(OTeg)]2-Aib-Ala-DMPE. ES-MS: m/zcalcd: 1623.01 [M + Na+], m/zfound: 1623.94 [M + Na+]; m/zcalcd: 801.00 [M/2 + H+], m/zfound: 801.51 [M/2 + H+]; m/zcalcd: 822.50 [M/2 + Na+]; m/ zfound: 822.99 [M/2 + Na+]. The synthesis of the thiolpeptide HS-(CH2)2-CO[Aib-Glu(OTeg)]2-Aib-Ala-OH, henceforth referred to as TP, was performed as described in [7]. 2.2. Chemicals and instrumentation The water used was obtained from water produced by an inverted osmosis unit by distilling it once and then by distilling the water so obtained from alkaline permanganate. Merck suprapur KCl was baked at 500 C before use to remove any organic impurities. DOPC and DMPE were obtained from Lipid Products (South Nutfield, Surrey, England). Gramicidin, alamethicin, melittin and cholesterol were purchased from Sigma and used without further purification. The other chemicals and solvents were commercially available and used as received. DOPC solutions were freshly prepared by diluting an appropriate amount of the stock solution of this

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phospholipid with pentane. Working solutions of 8 · 10
5 M TP in water + methanol 1:1 (v/v) and of 5 · 10
4 M TL in methanol were prepared by diluting, respectively, their 10
2 and 3.3 · 10
3 M stock solutions in methanol. Both stock and working solutions were stored at
18 C under argon, to prevent the oxidation of the thiols to disulfides. Stock solutions of 1.2 · 10
4 M gramicidin were prepared in ethanol, those of 1 mg/ ml alamethicin in water + ethanol 9:1 (v/v) (all solutions were stored at +4 C). Stock solutions of melittin in water (100 lg/ml) were freshly prepared before use. All measurements were carried out in aqueous 0.1 M KCl. A homemade HMDE, described elsewhere [22], was employed. It allows accurate changes in the drop area of as little as 0.04 mm2. Use was made of a home-made glass capillary with a finely tapered tip, about 1 mm in outer diameter. The capillary and mercury reservoir were thermostated at 25 ± 0.1 C by the use of a water-jacketed box, to avoid any changes in drop area due to a change in temperature. Two glass electrolysis cells containing aqueous 0.1 M KCl and a small glass vessel containing the working solution of TP or TL were placed on a movable support inside the box [23]. The HMDE and the support were moved vertically and horizontally, respectively, by means of two oleodynamic systems that ensured the complete absence of vibrations. Monolayers of TP or TL were self-assembled on the HMDE by keeping the mercury drop immersed in the small vessel containing their solution for about 30 min. In the meantime, a pentane solution of DOPC or DOPC + cholesterol (10:1.8 mol/mol) was spread on the surface of the 0.1 M KCl aqueous solution contained in one of the two electrolysis cells, in an amount corresponding to five to six phospholipid monolayers, and the pentane was allowed to evaporate. Using the oleodynamic system, the mercury drop coated with the TP or TL monolayer was then extracted from the vessel, rinsed with the organic solvent to remove the excess of the adsorbed thiol and kept in an argon atmosphere for the time strictly necessary to allow the solvent to evaporate. Immediately afterwards, the electrolysis cell containing the aqueous solution on the surface of which the phospholipid had been previously spread was brought below the HMDE, which was lowered so as to bring it into contact with the phospholipid film. After positioning the drop in such a way as to keep the drop neck in contact with the lipid reservoir [6,7], the applied potential was repeatedly scanned between
0.3 and
0.9 V(SCE) for TP-coated mercury, and from
0.3 to
1.7 V(SCE) for TL-coated mercury, while continuously monitoring the curve of the differential capacity, C, vs. the applied potential, E, until a stable C vs. E curve was attained. Measurements at a TP/DOPCcoated mercury drop were carried out by maintaining

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its neck in contact with the lipid reservoir. Conversely, measurements at a TL/DOPC-coated mercury drop were carried out after immersing it completely into the electrolyte solution, scanning the applied potential from
0.4 to
0.8 V(SCE) and transferring it into the cell containing the 0.1 M KCl aqueous solution with no lipid film on its surface. The impedance spectra of the TPcoated HMDE were recorded by transferring the electrode from the TP containing vessel in the KCl aqueous solution, in the absence of the DOPC film on its surface; the applied potential was then scanned from
0.2 to
1.7 V(SCE) for 20–30 min, until a stable differential capacity vs. potential curve was attained. To incorporate gramicidin, alamethicin and melittin, their stock solutions were simply added to the aqueous subphase and stirred for a few minutes, while keeping the Hg-supported lipid bilayer at an applied potential lying over the minimum capacitance region. The reproducibility and stability of the bilayer were tested before any addition. Ac voltammetry and impedance spectroscopy measurements were carried out with an Autolab instrument (Echo Chemie) supplied with an FRA2 module for impedance measurements, SCAN-GEN scan generator and GPES 4.9 software. The fitting routine supplied by the instrument is based on the program EQUIVCRT by B.A. Boukamp. Potentials were measured versus a Ag|AgCl (0.1 M KCl) reference electrode, but are referred to a saturated calomel electrode (SCE). The resistance and capacity of the Hg-supported biomimetic membranes were estimated from electrochemical impedance spectra covering the frequency range from 0.1 to 105 Hz, at an applied potential lying over the minimum capacitance region.

3. Results and discussion In spite of some satisfactory results obtained with lipid bilayers self-assembled on top of mercury-supported hydrophilic spacers such as triethyleneoxythiol [6] and TP [7], their use is somewhat awkward, since the drop neck must be kept in contact with the lipid reservoir on the surface of the aqueous electrolyte during measurements. In fact, their complete immersion into the aqueous solution destabilizes them because of the non negligible solubility of the hydrophilic spacer. This inconvenience is removed with the thiolipid TL, since its solubility in water is notably decreased by the phospholipid moiety. Thus, a mercury-supported lipid bilayer formed with TL is entirely stable when immersed in an aqueous solution, and can also be safely transferred from one solution to another. Fig. 2 shows the logarithm, log|Z|, of the modulus of the impedance and the phase angle of a biomimetic membrane formed by self-assembling a DOPC mono-

Fig. 2. Logarithm of the modulus of the impedance of TL/DOPCcoated Hg (solid circles) and of TL/(DOPC-Chl)-coated Hg (solid triangles) and phase angle of TL/DOPC-coated Hg (open circles), against the logarithm of the frequency, at
0.550 V. The phase angle of TL/(DOPC-Chl)-coated Hg was not reported because it almost coincides with that of TL/DOPC-coated Hg. The solid curves are leastsquares fits to an equivalent circuit consisting of an RC mesh with a resistance RX in series with it: C = 2.0 lF cm
2, R = 0.2 MX cm2 and RX = 160 X for TL/DOPC-coated Hg; C = 1.3 lF cm
2, R = 0.1 MX cm2 and RX = 160 X for TL/(DOPC-Chl)-coated Hg.

layer on top of a TL-coated mercury, against the logarithm of the frequency at
0.550 V. The impedance spectrum varies only slightly over the potential range from
0.450 and
0.800 V. The solid curve in Fig. 2 is the best fit of the impedance spectrum to an equivalent circuit consisting of an RC mesh simulating the lipid bilayer, with in series the resistance RX of the aqueous solution. The fit yields a C value of 2 lF cm
2 and an R value of 0.2 MX cm2. The differential capacity of the lipid bilayer is appreciably higher than that, 0.8, of a solvent-free black lipid membrane, BLM [24]. This finding denotes a poor organization of the lipid bilayer, which is not observed when a whole lipid bilayer self-assembles on top of a TP-coated mercury drop [7]. This behavior is probably to be ascribed to the fact that the cross sectional area of the TL hexapeptide moiety, with its two triethyleneoxy side chains, is greater than that of the DMPE moiety. To form a compact lipid bilayer tethered to the mercury surface, the empty space in the lipid monolayer covalently linked to the hexapeptide moiety should be filled with additional lipid molecules, when self-assembling the second lipid monolayer on top of the TL monolayer. Apparently, the bent, unsaturated oleoyl chains of DOPC do not intercalate satisfactorily into the straight, saturated myristoyl chains of the thiolipid. Better results were obtained by self-assembling the second monolayer from a 10:1.8 (mol/mol) DOPC + cholesterol mixture spread on the surface of the aqueous

L. Becucci et al. / Journal of Electroanalytical Chemistry 576 (2005) 121–128

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electrolyte. The presence of cholesterol decreases C down to about 1.3 lF cm
2 over a potential range of about 400 mV. The decrease in capacity upon addition of cholesterol is responsible for the shift of the Bode plot towards higher frequencies, as shown in Fig. 2. The above supported membrane allows the incorporation of both gramicidin and alamethicin from their very dilute aqueous solutions. A further increase in the percentage of cholesterol, while causing an additional decrease in capacity, was found to increase the rigidity of the lipid bilayer to such an extent as to prevent any reconstitution. The results reported in what follows were obtained by self-assembling the second lipid monolayer from the 10:1.8 (mol/mol) DOPC + cholesterol mixture.

about +250 mV. Consequently, the transmembrane potential equals zero at an absolute potential difference w of about
0.480 V. When the sulfydryl groups of the thiolipid bind to the metal surface, they transfer a fraction,
keN, of their total negative charge to the metal, where N is the number density of the chemisorbed TL molecules and k is the partial charge transfer coefficient. Incidentally, in the case of a Hg-supported TP monolayer, k was found to be practically equal to unity [7], thus denoting total charge transfer; this conclusion can be reasonably extended to the TL monolayer. In the case of partial or total charge transfer, w is approximately expressed by the equation [19]:     q q þ F Cþ w¼ þ vs þ þ vm þ w d : ð1Þ Cs Cm

3.1. Incorporation of gramicidin

Here Cs and vs are the capacity and dipole potential of the hydrophilic moiety, respectively, while Cm and vm are the corresponding quantities for the lipid bilayer; wd is the potential difference across the diffuse layer located on the solution side of the lipid bilayer; q is the ‘‘free charge density’’ on the mercury surface, namely the charge density experienced by the diffuse-layer ions; FC+ is the charge density of the K+ ions present in the hydrophilic spacer, assumed to be located at the boundary between the hydrophilic spacer and the lipid bilayer, for simplicity. The free charge density q is given by the experimentally accessible charge density, rM, that flows along the external circuit at constant applied potential when the electrode surface area is increased by unity, plus the negative charge density,
keN, transferred from the sulfur atoms to the metal. The first expression between round brackets in Eq. (1) is the potential difference across the hydrophilic moiety, while the second expression is the transmembrane potential. The dipole potential located in the polar head of the DMPE moiety of the thiolipid can be reasonably regarded as approximately equal in magnitude to that located in the polar head of the adjacent DOPC monolayer, but with an opposite orientation. Hence, the dipole potential, vm, which is the sum of the two, can be regarded as negligibly small. Consequently, at zero transmembrane potential, the overall charge, q + FC+, on the metal side of the lipid bilayer is vanishingly small, and the same is true on its solution side, to ensure the electroneutrality of the whole metal|solution interphase. This implies that the potential difference wd across the diffuse layer is also equal to zero. From Eq. (1) it follows that at zero transmembrane potential, when w is equal to
0.480 V, it is simply given by the expression (q/Cs + vs). In practice, under these conditions, q is also very close to zero (see, e.g., Fig. 2 in [19]) because the two contributions to q, rM and
keN are almost equal in magnitude and opposite in sign. The dipole potential, vs, of the hydrophilic spacer can, therefore, be roughly estimated at
480 mV.

Fig. 3 shows the conductance of a Hg-supported TL/ (DOPC-Chl) film incorporating gramicidin against the applied potential E, as measured by the in-phase component, Y 0 , of the electrode admittance at 10 Hz. The inflection point of the sigmoidal curve of Y 0 vs. E lies at about
0.730 V(SCE). It was shown elsewhere [19] that this potential can be roughly regarded as the applied potential at which the potential difference across the lipid bilayer (the transmembrane potential) vanishes. It was also shown that, apart from a small and almost potential-independent contribution due to electron spillover from the metal, the absolute potential difference, w, across the interphase between mercury and an aqueous solution can be estimated approximately by increasing the applied potential E measured versus SCE by

Fig. 3. Plot of the in-phase component, Y 0 , of the electrode admittance at 10 Hz against E for TL/(DOPC-Chl)-coated Hg in aqueous 0.1 M KCl, both in the absence (open circles) and in the presence of gramicidin incorporated from its 2 · 10
7 M solution (solid circles).

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L. Becucci et al. / Journal of Electroanalytical Chemistry 576 (2005) 121–128

This vs value was confirmed by measuring the free charge density q experienced by the diffuse-layer ions, as estimated on the basis of the Gouy–Chapman (GC) theory by varying the KCl concentration of the aqueous solution [7]. These measurements could be carried out only at a mercury electrode coated with a monolayer of the sole hydrophilic spacer, TP, the capacity of which, Cs, is about one order of magnitude greater than that of the TL/(DOPC-Chl) film. Indeed, the capacity of the latter film is so low as to obscure completely the much higher diffuse-layer capacity, Cd, in series with it. Impedance spectra at
1.000 V of a mercury electrode coated with the thiolpeptide TP and immersed in KCl solutions of concentration c ranging from 5 · 10
3 to 0.1 M were fitted to an equivalent circuit consisting of a RsCs mesh representing the hydrophilic spacer, with, in series, a further RdCd mesh, representing the diffuse layer; in the fitting, the elements of the RsCs mesh were forced to remain constant with varying KCl concentration [7]. The resulting Cs value equals 11 lF cm
2, while Rs amounts to 0.17 MX cm2. Fig. 4 shows the reciprocal, 1/Cd, of the experimental diffuse-layer capacitance against the l/Cd (rM = 0) value corresponding to the same KCl concentration, as calculated on the basis of the GC theory. The figure also shows 1/Cd(rM) vs. 1/ Cd(rM = 0) plots calculated from the GC theory for different values of the charge density rM on the metal. The experimental points are in fairly good agreement with the 1/Cd(rM) vs. 1/Cd(rM = 0) plot corresponding to rM =
3 lC cm
2. It must be stressed that, in the present case, the charge of
3 lC cm
2 is just the extrathermodynamic charge, q, experienced by the diffuse-layer ions, and not the thermodynamically significant charge density, rM, on the metal, because of partial charge

transfer from sulfur to mercury. At the applied potential of
1.000 V, the potential difference w across the whole interphase equals (
1.000 + 0.250) V =
0.750 V. In the absence of the lipid bilayer on top of the hydrophilic spacer, Eq. (1) reduces to: w ¼ ðq=C s Þ þ vs þ wd :

ð2Þ


2

For q =
3.0 lC cm and c = 0.1 M KCl, the GC theory affords a wd value of
38 mV. Replacing this wd value, w =
0.750 V, q =
3.0 lC cm
2, and Cs = 11 lF cm
2 in Eq. (2), a vs value of
0.440 V is obtained, in fairly good agreement with the value obtained from Fig. 2. 3.2. Incorporation of alamethicin Fig. 5 shows the in-phase component, Y 0 , of the electrode admittance at 10 Hz against the applied potential E at a Hg-supported TL/(DOPC-Chl) film immersed in 0.1 M KCl aqueous solutions containing different concentrations of alamethicin. Alamethicin is the best known member of the class of fungal antibiotics known as peptaibols, which are characterized by several Aib residues [25–27]. This non-coded Ca-tetrasubstituted aamino acid residue is considered to favor the formation of helical structures [28]. Alamethicin exhibits a strongly voltage-dependent conductance in BLMs. If BLMs are formed from DOPC, addition of alamethicin to one side produces an increase in electrical conductance when voltages of either polarity are applied; the conductance increase over the bare membrane level occurs at a somewhat lower voltage when the side opposite to that where the alamethicin is added is negative [29]. With BLMs formed with saturated phospholipids, no con-

0.03 σM=-1 µC cm-2 σ =-1.5 M

0.025

σ =-2

Cd-1 / cm2 µF-1

M

σM=-2.5

0.02

σM=-3 0.015

σM=-4

0.01 0.005

0.0

0.01

0.02

0.04

0.03

Cd (σM=0) / cm µF -1

2

0.05

0.06

0.07

-1

Fig. 4. Reciprocal, 1/Cd, of the experimental diffuse-layer capacity of TP-coated Hg immersed in 5 · 10
3 M, l.3 · 10
2 M, 3.6 · 10
2 M and 0.1 M KCl at
1.000 V. against the 1/Cd(rM = 0) value corresponding to the same KCl concentration, as calculated on the basis of the Gouy–Chapman (GC) theory. The solid curves are 1/Cd(rM) vs. 1/Cd(rM = 0) plots calculated from the GC theory for different charge densities rM on the metal, the values of which are reported on each curve.

L. Becucci et al. / Journal of Electroanalytical Chemistry 576 (2005) 121–128 2 10-4 d

1.5 10 -4 G/Mho cm-2

c

1 10-4

b

a

5 10

-5

0

0 10

-0.9

-0.8

-0.7

-0.6

-0.5

-0.4

-0.3

E/V (SCE)

Fig. 5. Plot of the in-phase component, Y 0 , of the electrode admittance at 100 Hz against E for TL/(DOPC-Chl)-coated Hg in aqueous 0.1 M KCl, both in the absence (curve a) and in the presence of alamethicin incorporated from its 1 (curve b), 2 (curve c) and 3 lg/ml (curve d) solution.

ductance increase was reported when this side is positive. The conductance is ascribed to the highly polar helical monomers spanning the membrane under the influence of an electric field and aggregating to form pores with different diameters. These pores are not very ion specific. Alamethicin conductance in BLMs increases exponentially with the applied voltage and is proportional to its bulk concentration raised to a power close to 10 [29]. The latter concentration dependence has suggested that an average of 10 alamethicin monomers aggregate to form the pore. The behavior of alamethicin incorporated in the Hgsupported TL/(DOPC-Chl) film is quite different from that in BLMs. Thus, the conductance increases progressively towards more negative potentials, but tends to level off, giving rise to roughly sigmoidal curves. This behavior may possibly be ascribed to the hydrophilic moiety of the thiolipid starting to be saturated with inorganic ions. The height of the plateau of the sigmoidal curves is roughly proportional to the alamethicin concentration. This weak concentration dependence may be due to some difficulty of the DMPE moiety of the thiolipid molecules tethered to the Hg surface to accommodate clusters of increasing size with an increase in alamethicin bulk concentration, Still more puzzling behavior is represented by the conductance starting to increase over the background value when the applied potential is still about 200 mV more positive than the value,
0.730 V(SCE), corresponding to a zero transmembrane potential. For comparison, melittin, another channel-forming peptide, was incorporated in the present Hg-supported membrane. Melittin, the main toxin of bee venom, is a natural peptide not containing Aib res-

127

idues [30]; it shares with alamethicin the capability of spanning a lipid bilayer when the transmembrane potential is made sufficiently negative on the side opposite to that where the peptide is present, forming pores by aggregation of monomers. No appreciable increase in conductance over the background value was observed throughout the whole potential range of stability of the biomimetic membrane by incorporating melittin from its 1.2 lg/ml aqueous solution (data not shown). A possible explanation for this apparently anomalous behavior may lie in the affinity of alamethicin with the hexapeptide chain of the thiolipid; thus, both peptides are characterized by the presence of Aib residues favoring 310-helical structures via intramolecular H-bond formation. This ‘‘3D-structural’’ affinity may overcome an electric field opposing the insertion of alamethicin into the lipid bilayer with its N-terminus, provided the field is not too high. In conclusion, the present thiolipid tethered to a mercury electrode, with a self-assembled monolayer on top, constitutes a valid biomimetic membrane. Its response to the incorporation of the ion channel gramicidin and its impedance spectrum indicates that this membrane model has a good fluidity and consists of a stable lipid bilayer on top of a hydrophilic spacer acting as an ionic reservoir.

Acknowledgments The authors are grateful to MIUR (Ministry of Education, University and Research) of Italy for financial support through the Grant PRIN 2003 035241 and to Ente Cassa di Risparmio di Firenze for financial support through the PROMELAB project.

References [1] R. Guidelli, G. Aloisi, L. Becucci, A. Dolfi, M.R. Moncelli, F. Tadini Buoninsegni, J. Electroanal. Chem. 504 (2001) 1. [2] L.M. Williams, S.D. Evans, T.M. Flynn, A. Marsh, P.F. Knowles, R.J. Bushby, N. Boden, Langmuir 13 (1997) 751. [3] L.M. Williams, S.D. Evans, T.M. Flynn, A. Marsh, P.F. Knowles, R.J. Bushby, N. Boden, Supramolecular Sci. 4 (1997) 513. [4] A. Toby, A. Jenkins, R.J. Bushby, N. Boden, S.D. Evans, P.F. Knowles, Q. Liu, R.E. Miles, S.D. Ogier, Langmuir 14 (1998) 4675. [5] C. Steinem, A. Janshoff, W.P. Ulrich, M. Sieber, H.-J. Galla, Biochim. Biophys. Acta 1279 (1996) 169. [6] L. Becucci, R. Guidelli, Q. Liu, R.J. Bushby, S.D. Evans, J. Phys. Chem. B 106 (2002) 10410. [7] C. Peggion, F. Formaggio, C. Toniolo, L. Becucci, M.R. Moncelli, R. Guidelli, Langmuir 17 (2001) 6585. [8] H. Lang, C. Duschl, H. Vogel, Langmuir 10 (1994) 197. [9] C. Duschl, M. Liley, G. Corradin, H. Vogel, Biophys. J. 67 (1994) 1229. [10] C. Steinem, A. Janshoff, K. von dem Bruch, K. Reihs, J. Goossens, H.-J. Galla, Bioelectrochem. Bioenerg. 45 (1998) 17.

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L. Becucci et al. / Journal of Electroanalytical Chemistry 576 (2005) 121–128

[11] B. Raguse, V. Braach-Maksvytis, B.A. Cornell, L.G. King, P.D.J. Osman, R.J. Pace, L. Wieczorek, Langmuir 14 (1998) 648. [12] B.A. Cornell, V.L.B. Braach-Maksvytis, L.G. King, P.D.J. Osman, B. Raguse, L. Wieczorek, R.J. Pace, Nature 387 (1997) 580. [13] R. Naumann, A. Jonczyk, R. Kopp, J. van Esch, H. Ringsdorf, W. Knoll, P. Gra¨ber, Angew. Chem. Int. Ed. Engl. 34 (1995) 2056. [14] R. Naumann, A. Jonczyk, C. Hampel, H. Ringsdorf, W. Knoll, N. Bunjes, P. Gra¨ber, Bioelectrochem. Bioenerg. 42 (1997) 241. [15] N. Bunjes, E.K. Schmidt, A. Jonczyk, F. Rippmann, D. Beyer, H. Ringsdorf, P. Gra¨ber, W. Knoll, R. Naumann, Langmuir 13 (1997) 6188. [16] S. Heyse, O.P. Ernst, Z. Dienes, K.P. Hofmann, H. Vogel, Biochemistry 37 (1998) 507. [17] E.K. Schmidt, T. Liebermann, M. Kreiter, A. Jonczyk, R. Naumann, A. Offenhausser, E. Neumann, A. Kukol, A. Maelicke, W. Knoll, Biosens. Bioelectron. 13 (1998) 858. [18] R. Naumann, E.K. Schmidt, A. Jonczyk, K. Fendler, B. Kadenbach, T. Liebermann, A. Offenha¨usser, W. Knoll, Biosens. Bioelectron. 14 (1999) 651. [19] L. Becucci, M.R. Moncelli, R. Guidelli, Langmuir 19 (2003) 3386.

[20] C. Toniolo, E. Benedetti, Trends Biochem. Sci. 16 (1991) 350. [21] F. Tadini Buoninsegni, L. Becucci, M.R. Moncelli, R. Guidelli, J. Electroanal. Chem. 500 (2001) 395. [22] M.R. Moncelli, L. Becucci, J. Electroanal. Chem. 433 (1997) 91. [23] F. Tadini Buoninsegni, R. Herrero, M.R. Moncelli, J. Electroanal. Chem. 452 (1998) 33. [24] M. Montal, P. Mueller, Proc. Natl. Acad. Sci. USA 69 (1972) 3561. [25] E. Benedetti, A. Bavoso, B. Di Blasio, V. Pavone, C. Pedone, C. Toniolo, G.M. Bonora, Proc. Natl. Acad. Sci. USA 79 (1982) 7951. [26] I. Vodyanoy, J.E. Hall, T.M. Balasubramanian, Biophys. J. 42 (1983) 71. [27] J.E. Hall, I. Vodyanoy, T.M. Balasubramanian, G.R. Marshall, Biophys. J. 45 (1984) 233. [28] C. Toniolo, M. Crisma, F. Formaggio, C. Peggion, Biopolymers (Pept. Sci.) 60 (2001) 396. [29] M. Eisenberg, J.E. Hall, C.A. Mead, J. Membr. Biol. 14 (1973) 143. [30] M. Pawlak, S. Stankowski, G. Schwarz, Biochim. Biophys. Acta 1062 (1991) 94.