Biophysical and Functional Characterization of an Ion Channel Peptide Confined in aSol-Gel Matrix

Rocıo Esquembre, Jose Antonio Poveda, and C. Reyes Mateo*Instituto de Biologıa Molecular y Celular. UniVersidad Miguel Hernandez de Elche,03202 Elche (Alicante), Spain

ReceiVed: March 3, 2009; ReVised Manuscript ReceiVed: April 15, 2009

Immobilization of zwitterionic lipid membranes in sol-gel matrices induces irreversible alterations of thebilayer fluidity, which can limit the use of these systems for practical applications. Recently, we have reportedthat electrostatic interactions between phospholipids polar heads and the negative-charged silica surface ofthe porous matrix should be the cause of such behavior. In the present work, we analyze the effect of theseinteractions on the biophysical and functional properties of the ion-channel peptide gramicidin, entrapped ina sol-gel matrix, to get more insight on the ability of these inorganic materials to immobilize ion channelsand other membrane-bound proteins. Gramicidin was reconstituted in anionic and zwitterionic liposomes andthe effects of sol-gel immobilization on the biophysical properties of gramicidin were determined from changesin the photophysical properties of its tryptophan residues. In addition, the physical state of the immobilizedlipid membrane containing gramicidin was analyzed by measuring the spectral shift of the fluorescent probeLaurdan. Finally, the ion-channel activity of the peptide was monitored upon sol-gel immobilization througha fluorescence quenching assay using the fluorescent dye pyrene-1,3,6,8-tetrasulfonic acid (PTSA). Resultsshow that the channel properties of the immobilized gramicidin are preserved in both zwitterionic and anionicliposomes, even though the zwitterionic polar heads interact with the porous surface of the host matrix.

Introduction

Ion channels are membrane proteins involved in the main-tenance of the appropriate ion balance across biologicalmembranes, connecting the inside of the cell to its outside in aselective fashion. They are natural nanotubes, which serve askey elements in signaling and sensing pathways and thus governan enormous range of biological functions important in healthand disease.1 The ability to immobilize these proteins ininorganic matrices represents a significant step forward indeveloping a new generation of biologically active materialswith potential applications in areas such as high-throughput drugscreening and new generation of biosensors.2 The majorlimitation in the development of these new materials involvesthe difficulty of finding immobilization techniques able to retainthe physical properties of the lipid bilayer where the ion-channelactivity is included. Because of their dynamic and self-assemblednature, fixation of lipid membranes to solid surfaces easilydisrupts the hydrophobic interactions that create lipid bilayersand modifies the natural dynamic motions of the membrane andits thermotropic properties, producing unstable immobilizedstructures and, in some cases, the rupture of the bilayer. Differentstrategies have been developed to overcome these difficulties.3

Among them, use of sol-gel materials seems to be an interestingalternative to immobilize lipid membranes vesicles (liposomes)and membrane-bound proteins in solid matrices without the needfor tethering the lipids to a surface.4,5 This sol-gel methodologyhas been extensively used to immobilize soluble proteinsshowing that the majority of them can be encapsulated withretention of their native structure and functionality and anenhanced stability.6-10 Bioencapsulation within these materialsis currently obtained from the hydrolysis of alkoxide precursors

(usually tetraethyl orthosilicate, TEOS, or tetrametyl orthosili-cate, TMOS) resulting in a colloidal sol solution. Subsequently,a buffered aqueous solution containing the biomolecule ofinterest is added to the sol producing a polycondensation reactionthat leads to the formation of a transparent highly porous gelthat encloses the species within their pores.

Yamanaka et al.11 and Nguyen et al.12 used, for the first time,the sol-gel methodology to immobilize liposomes for heavy-metal ion and pH sensing, respectively. A detailed characteriza-tion of the physical properties and stability of the immobilizedlipid systems was carried out later by the Brennan group.13 Theyincorporated liposomes composed of dipalmitoyl phosphatidyl-choline (DPPC) in a TEOS sol-gel matrix and observed thatthe lipid bilayer did not exhibit phase transition suggesting thatencapsulation resulted in rupture of these structures. Thisbehavior was attributed to interactions between the lipid andthe ethanol resulting as a byproduct of the chemical reactionsinvolved in the formation process of the silica matrix. A similarconclusion was reached by Halder et al.14 when encapsulatingdimyristoyl phosphatidylcholine (DMPC) in TEOS glasses. Toovercome these problems, use of either new precursors (otherthan TEOS and TMOS), which minimize the generatedalcohol,13,15 or alternative alcohol free routes16,17 is required.Nevertheless, it has been reported that, for pure zwitterionicliposomes, (i.e., DMPC, DPPC) the lipid phase transition isaffected although these routes are used, suggesting irreversiblealterations of the bilayer fluidity that could prevent the use ofthese systems for practical applications.13,18 Recently, we havereported that electrostatic interactions between phospholipids’polar heads and the negative-charged silica surface of the porousmatrix seem to play a capital role for the preservation of thestructural integrity of the immobilized bilayer.17 Here, weanalyze the effect of such interactions on the biophysical andfunctional properties of the model ion-channel gramicidin to

* To whom correspondence should be addressed. Tel.: +34 966 658469. Fax: +34 966 658 758. E-mail: rmateo@umh.es.

J. Phys. Chem. B 2009, 113, 7534–75407534

10.1021/jp9019443 CCC: $40.75 2009 American Chemical SocietyPublished on Web 05/04/2009

get more insight on the ability of these inorganic materials toimmobilize ion channels and other membrane-bound proteins.

Gramicidin is a pentadecapeptide antibiotic that inserts intothe lipid bilayer forming prototypical ion channels specific forthe transport of monovalent cations across membranes.19,20 Thispeptide has one of the most hydrophobic sequences known andis very sensitive to the environment in which it is placed,adopting a wide range of conformations, depending on the natureof solvent in which it is dissolved and on the incorporationmethod.19-21 In membranes, the most preferred conformationsare the single-stranded helical dimer (the channel form) and thedouble-stranded interwined helix (the nonchannel form), whichshow different fluorescent properties.20 Recently, the elucidationof the X-ray crystal structure of the Streptomyces liVidans K+

channel (KcsA) in molecular detail22 has allowed to verify thatthe gramicidin channel form shares important structural featureswith real protein ion channels.21,23 Consequently, this peptideappears as a useful model for realistic determination of theeffects of sol-gel immobilization on the biophysical andfunctional properties of membrane-bound proteins.

Successful immobilization of the gramicidin channel formin sol-gel materials was reported for the first time by Brennan’sgroup using as silica precursor a diglycerilsilane derivativesynthesized by themselves.24 However, when they tried to entrapthis ionic channel using the traditional silica precursors TMOSor TEOS, results were unsuccessful, probably due to the above-mentioned damaging effects of the alcohol generated during thehydrolysis process. Recently, in a preliminary work we managedto immobilize gramicidin in a TMOS sol-gel matrix using analcohol-free route developed by Ferrer et al.,25 obtaining asystem that was able to generate ion fluxes.18 In the presentwork, we have used the same methodology to immobilizegramicidin reconstituted in zwitterionic and anionic liposomes,and the effects of the phospholipids polar heads on thebiophysical and functional properties of the immobilized peptidehave been analyzed through steady-state and time-resolvedfluorescence techniques. Fluorescence spectra, quenching ex-periments, and fluorescence lifetimes of the gramicidin tryp-tophans indicate that the biophysical properties of the recon-stituted ionic channel are preserved in both zwitterionic andanionic liposomes upon sol-gel immobilization, but the lipidbilayer physical state, studied by the fluorescent probe Laurdan,is altered only in the case of zwitterionic phospholipids. Ion-channel activity of the immobilized gramicidin was monitoredthrough a fluorescence quenching assay using the fluorescentdye pyrene-1,3,6,8-tetrasulfonic acid (PTSA). Results show that,contrary to that expected, the ionic channel activity is preservedin both zwitterionic and anionic liposomes, even though thezwitterionic polar heads seem to interact with the porous surfaceof the host matrix.

Materials and Methods

Chemicals. The precursor tetramethyl orthosilicate (TMOS)and the phospholipids phosphatidylglycerol (EyPG) and phos-phatidylcholine (EyPC), both derived from egg yolk, werepurchased from Sigma-Aldrich Chemical Co. (Milwauke, WI,USA). Gramicidin A′ and the fluorescent probes 2-dimethy-lamino-6-lauroylnaphtalene (Laurdan) and 1,3,6,8-pyrene tet-rasulfonate (PTSA) were from Molecular Probes (Eugene, OR,USA) and used without further purification. Gramicidin A′, aspurchased, is a mixture of gramicidins A, B, and C. Water wastwice-distilled in all-glass apparatus and deionized using Milli-Qequipment (Millipore, Madrid, ES). Ethanol, methanol, and

chloroform (spectroscopic grade) were from Merck. All othercompounds were of analytical grade.

Liposome Formation and Gramicidin Reconstitution.Chloroform/methanol solutions containing 3 mg of total phos-pholipid (EyPC or EyPG) were dried first by evaporation undera dry nitrogen gas stream and subsequently under vacuum for3 h. Multilamellar vesicles (MLVs) were formed by resuspend-ing the dried phospholipid in buffer (Tris-HCl 50 mM, NaCl250 mM, pH 7.4) to a final concentration of 1 mM. The vesiclesuspension was vortexed several times. Large unilamellarvesicles (LUVs) with a mean diameter of 90 nm were preparedfrom these MLVs by pressure extrusion through 0.1 µmpolycarbonate filters (Nucleopore, Cambridge, MA).

Gramicidin A’ was incorporated into MLVs as describedpreviously.26 Briefly, a peptide/lipid mixture (1:60 molar ratio)was codissolved in methanol with a few drops of chloroform,dried first by evaporation under dry nitrogen gas stream andafter under vacuum, hydrated with 50 mM Tris-HCl buffer (pH7.4, 250 mM NaCl), and vortexed to obtain MLVs and,subsequently, LUVs as described above. The sample wasincubated overnight at 65 °C with continuous stirring to inducethe channel-forming �-helical monomeric conformation.27,28

Labeling of Liposomes. A few microliters from a stocksolution of the fluorescent probe Laurdan in ethanol was addedto the LUVs’ suspension. Solutions were incubated for 30 minat 30 °C to facilitate the probe incorporation into the lipidbilayer. The ethanol final concentration was always less than2%. The lipid-to-probe ratio, in molar terms, was 500:1.

For samples to be used in the ion-channel activity assay, thefluorescent dye PTSA was encapsulated into the aqueous interiorof LUVs formed as described above, except that the driedphospholipid was resuspended in a buffer containing PTSA 4.16mM. Removal of the external fluorophore was accomplishedby chromatography on a sepharose G-50 column (15 × 0.9 cm)using buffer Tris-HCl 50 mM pH 7.4, NaCl 250 mM as eluant.

Immobilization of Liposomes in Sol-Gel Monoliths.LUVs, with or without gramicidin, were encapsulated into puresilica matrices using an alcohol-free route developed by Ferreret al.25 Silica sol stock solution was prepared by mixing 5.88mL of TMOS, 2.88 mL of H2O, and 0.06 mL HCl (0.62 M)under vigorous stirring at 4 °C in a closed vessel. After 50 min,1 mL of the resulting sol was mixed with 1 mL of deionizedwater and submitted to rotaevaporation for a weight loss of ∼0.6g (i.e., 0.6 g are approximately the alcohol mass resulting fromalkoxyde hydrolysis). The aqueous sol was mixed with 1 mLof a diluted buffered suspension of liposomes in a disposablecuvette of polymethylmethacrylate. Gelation occurs readily aftermixing. Afterward, monoliths (9 mm × 9 mm × 17 mm) werewet aged in a Tris buffer (50 mM, pH 7.5, NaCl 250 mM)solution at 4 °C before use.

Steady-State Fluorescence Measurements. Fluorescencemeasurements of gramicidin were performed in a SLM-8000Cspectrofluorimeter (SLM Instruments Inc., Urbana, IL, USA).Emission spectra were excited at 290 nm with emission collectedfrom 305 to 400 nm. The experimental samples (sol-gelmonoliths and lipid suspensions) were placed in 10 × 10 mmcuvettes. Background intensities due to the sol-gel matrix and/or liposomes were always taken into account and subtractedfrom the sample.

Fluorescence emission spectra of Laurdan were obtained witha Cary Eclipse spectrofluorometer (Varian) interfaced with aPeltier cell. Emission spectra were recorded at 25 °C, fixingexcitation wavelength at 350 nm. The spectral changes of the

Characterization of an Ion Channel Peptide J. Phys. Chem. B, Vol. 113, No. 21, 2009 7535

emission spectrum of the probe were also quantified by the so-called generalized polarization (GPex),29,30 which is defined aseq 1:

GPex )I440 - I490

I440 + I490(1)

where I440 and I490 are the fluorescence intensities recorded aftersubtraction of background intensity at the characteristic emissionwavelengths of the gel phase (440 nm) and of the fluid phase(490 nm). GPex values were obtained in a range of excitationwavelengths (325-410 nm) at 25 °C.

Time-Resolved Fluorescence Measurements. The decay ofthe total fluorescence intensity was recorded at 25 °C using aPTI model C-720 fluorescence lifetime instrument (PhotonTechnology International Inc., Lawrenceville, NJ) utilizing aproprietary stroboscopic detection technique.31 The systememploys a PTI GL-330 pulsed nitrogen laser pumping a PTIGL-302 high-resolution dye laser. The dye laser output at 594nm was frequency-doubled to 297 nm with a GL-303 frequencydoubler coupled to an MP-1 sample compartment via fiberoptics. The emission was observed at 90° relative to theexcitation via an M-101 emission monochromator and astroboscopic detector equipped with a Hamamatsu 1527photomultiplier.

The kinetic parameters of the impulse response fluorescenceintensity decay i(t) ) ∑i Ri exp (- t/τi), lifetimes τi, andnormalized amplitudes Ri, were recovered with the Felix 32analysis package using a discrete one- to four-exponential fittingprogram. The amplitude-weighted lifetime proportional toquantum yield, τj, and the average fluorescence lifetime, ⟨τ⟩, werecalculated according to τj ) ∑Riτi and ⟨τ⟩ ) (∑i Riτi

2)/(∑i Riτi),respectively.32 The fits tabulated represent the minimum set ofadjustable parameters that satisfy the usual statistical criteria,namely a reduced �2 value of <1.3 and a random distributionof weighted residuals.

Quenching Experiments. The accessibility to solvent of thegramicidin fluorescent tryptophans in both solution and sol-gel-entrapped liposomes of EyPG and EyPC was analyzed bymonitoring the fluorescence quenching of gramicidin inducedby adding increasing amounts of a 6.0 M acrylamide stocksolution with continuous stirring. Fluorescence intensity wascorrected from sample dilution and recorded at 340 nm (λexc )295 nm). Corrections for inner filter effect were also made asis described in Rawat et al.20 The absorbances of the sampleswere measured using a Shimadzu spectrophotometer (UV-1603,Tokio, Japan). For the quenching studies of the entrappedgramicidin/lipid bilayer, the diffusion constraints imposed bythe dimensions of the monoliths and the restriction to the freediffusion of the quencher through the sol-gel matrix requiredits incubation with the quencher solution for at least 48 h beforeconcentration gradients could be neglected. A different monolithcontaining the same gramicidin concentration was incubatedwith each quenching concentration. Data were analyzed ac-cording to the Stern-Volmer relationship:

I0

I) 1 + KSV[Q] (2)

where I0 and I stand for the steady-state fluorescence intensitiesin the absence and in the presence of quencher respectively,and [Q] is the quencher concentration. Because no significantdeviation was detected in these plots, the static quenchingcontribution was considered to account for less than 10% ofthe observed signal, and the Stern-Volmer constant, KSV, wasdirectly obtained from the slope of the linear relationship.33 This

parameter represents the weighted sum of the individualconstants of the emitting tryptophan residues and is thereforereferred to as KSV,eff in the text. KSV,eff was used to calculate thebimolecular rate constant of the quenching process, kQ,eff, fromthe expression KSV,eff ) kQ,eff ⟨τ⟩.

Ion-Channel Activity Assay. The activity of the sol-gelimmobilized gramicidin was analyzed by measuring the influxof Cs+ through this ion channel incorporated into LUVspreviously loaded with PTSA, a pyrene-derived water-solublefluorophore. This assay is based on the collisional quenchingof the entrapped fluorophore when externally added Cs+ reachesthe vesicle lumen through the gramicidin ion channel. Fluores-cence was recorded in the SLM-8000C spectrofluorimeter at386 nm and monitored as a function of CsCl concentration withan excitation wavelength of 355 nm. Because of the slowdiffusion of Cs+ thorough the sol-gel matrix, fluorescence wasmonitored periodically until it reached a stable value, whichwas used for the calculations. Buffer inside vesicles was Tris-HCl 50 mM, pH 7.5, NaCl 250 mM, whereas buffer outsidewas the same but substituting a certain amount of NaCl withCsCl so the total salt concentration remains 250 mM. The smalldrop in the PTSA fluorescence due to the passive leakage ofCs+ into the vesicles was corrected using samples with nogramicidin. To do so, as there are small variations in thefluorescence signal between every sol-gel monolith, possiblydue to little differences in scatter and/or sample content,fluorescence was measured initially in NaCl 250 mM bufferfor every monolith, and then washed with the correspondingbuffer containing CsCl, so measurements with and withoutgramicidin could be normalized to the initial value without anyquenching and then subtracted.

Results and Discussion

Effect of Sol-Gel on the Photophysical Properties ofMembrane-Bound Gramicidin. Most of the intrinsic fluores-cence of gramicidin comes from its tryptophan residues, whichare crucial for maintaining the structure and function of thechannel and have been shown to be localized at the membraneinterfacial region.20,34,35 Incorporation of gramicidin in LUVsof EyPC and EyPG and subsequent immobilization in a sol-gelmatrix was, therefore, monitored via tryptophan emission spectra(Figure 1). For gramicidin in buffer, a fluorescence maximumwas obtained around 339 nm, which was preserved upon sol-gelimmobilization (note that solubility of gramicidin in aqueous

Figure 1. Normalized fluorescence emission spectra of gramicidin insolution ( · · · ), (+) and in liposomes of EyPG (•), (s) and EyPC (O),(---) before and after sol-gel entrapment, respectively; T ) 25 °C,λexc ) 290 nm.

7536 J. Phys. Chem. B, Vol. 113, No. 21, 2009 Esquembre et al.

media is limited and leads to the growth of aggregates asindicated by an increase in light scattering of the sample36). Forthe peptide in EyPC, the emission spectrum shifted to 332 nm,due to a decrease in polarity of the surrounding environment.A similar behavior was reported by Chattopadhyay’s group forgramicidin incorporated in POPC, and was correlated with acorrect insertion of the peptide in the membrane, adopting thechannel conformation.20 For the peptide in EyPG, the emissionspectrum showed a further blue shift of 4 nm. This shift suggestsdifferences in the microenvironment experienced by the tryp-tophans of gramicidin bound to membranes of different chargetypes, which could be due to a deeper location of gramicidin inthe negatively charged bilayer and/or a lower interfacial watercontent in the membrane interior as was proposed by othersgroups for the membrane-bound melittin peptide.37,38 Indepen-dently of these differences, it was observed that, for both typeof lipids, fluorescence emission spectra did not change aftersol-gel entrapment (Figure 1 and Table 1) indicating thatgramicidin/liposomes composed either by zwitterionic or byanionic phospholipids are successfully encapsulated in thesol-gel glass and that the microenvironment surrounded tryp-tophans within the sol-gel pore seems to be similar to that oneobserved for the liposomes freely suspended in water. Toconfirm this observation, red edge excitation shift (REES)experiments were carried out. The phenomenon of REES hasbeen widely studied for gramicidin inserted in membranes byChattopadhyay’s group and used (with related fluorescenceapproaches) to characterize different conformations adopted bythe peptide in the membrane.20,26 Nevertheless, in our case noresults could be drawn due to low signal-to-noise ratio andartifacts at long excitation wavelengths caused by turbidity ofsol-gel samples that remained even after background subtraction.

Further information regarding the environment of the grami-cidin tryptophans in free and sol-gel entrapped liposomes wasobtained from the analysis of their fluorescence emission decay.Experimental fluorescence decays were best fitted to biexpo-nential functions. This biexponential behavior has been similarlyreported for gramicidin reconstituted in other phospholipidssystems.20,21 Fluorescence fit parameters, including τj and ⟨τ⟩,are shown in Table 1. A definite assignment of the observeddiscrete lifetime components to the different fluorescent Trpresidues is difficult for multitryptophan proteins or peptides likegramicidin. In this case, it is more useful to compare the averagefluorescence lifetimes ⟨τ⟩. For gramicidin incorporated in LUVsof EyPC freely suspended in buffer, this value was found to besimilar to that reported in other phosphatidylcholine bilayersfor the peptide in the channel form and was clearly shorter thanthat corresponding to the nonchannel conformation.20,21 Im-mobilization of the zwitterionic system in the sol-gel matrixscarcely affected the fluorescence lifetimes of the peptideindicating that gramicidin not only remains within the lipidbilayer during the immobilization process but also that the

channel conformation seems to be preserved. For gramicidinin EyPG, the fluorescence lifetimes were also similar for thefree and the immobilized system, with values also in agreementto those for the peptide in the channel form, but shorter thanthose recovered for the zwitterionic lipid. This result wassurprising because there is generally a correlation between ablue shift of the wavelength of maximum emission in tryptophanproteins and an increase in their fluorescence lifetime. In thissense, we expected that, if there were a shallower location ofgramicidin and/or a higher interfacial water content in zwitte-rionic than in anionic membranes then the fluorescence lifetimeof gramicidin should be shorter in EyPC than in EyPG becauseof the strong quenching effect of water molecules.39 Theseresults suggest that the conformation of gramicidin into theanionic membrane bilayer is slightly different to that adoptedin the zwitterionic system. This hypothesis is supported by datafrom Marsh’s group, which reported that gramicidin insertedin anionic lipids shows a lower degree of orientation relativeto the lipid acyl chains than that in DMPC.40 In this newsituation, which is preserved in the sol-gel matrix, gramicidincould suffer an intramolecular quenching process between thepeptide backbone and some of its tryptophans, which wouldexplain the shortening of its lifetime. A similar effect butcomprising a quenching from a lysine residue was invoked toexplain the shortening of lifetime in the mellitin peptide whenincorporated into DOPC membranes.37 In any case, thesechanges should not be very drastic because, as can be inferredfrom the activity assays reported by Rostovstera et al.41 and byourselves in this work (results below), the channel conformationof the peptide is preserved upon insertion in the negativelycharged membrane.

Quenching Experiments. Additional information regardingthe effect of sol-gel encapsulation on the gramicidin channelcan also be inferred from fluorescence quenching experiments.For this purpose, the effect of acrylamide on the intrinsicfluorescence of entrapped gramicidin was examined and com-pared with that observed for the liposomes freely suspended inwater. For multitryptophan peptides such as gramicidin, deter-mination of quenching constants would be ambiguous withoutselective replacement of Trp residues by nonfluorescent residues.However, in this work we did not pursue a rigorous quantitativeinterpretation of the quenching data limiting ourselves tocomparing changes observed in the average fluorescencequenching efficiency obtained for the peptide incorporated inzwitterionic and anionic lipids before and after sol-gel encap-sulation. With this in mind, experimental data were analyzedin terms of eq 2. The Stern-Volmer plots were linear (Figure2), and the KSV,eff values extracted from the slopes, whichtogether with the average fluorescence lifetimes ⟨τ⟩ reported inTable 1, were used to calculate the bimolecular rate constantof the quenching process, kQ,eff (Table 2). In solution, the kQ,eff

value recovered for the anionic system was slightly lower thanthat obtained for the zwitterionic one, indicating that accessibilityof acrylamide to the fluorescent tryptophans is lower in EyPGthan that in EyPC, probably due to the differences in theinsertion mode of the peptide in both membranes, which shouldimply a deeper location of gramicidin and/or a smaller interfacialwater content in the anionic bilayer. This result is in agreementwith the blue-shift in the fluorescence emission spectrumreported for the peptide in EyPG. Encapsulation of this lastsystem within the sol-gel matrix did not affect the value ofkQ,eff, indicating that the accessibility of the quencher to thetryptophans remains essentially unaltered upon immobilizationand that, therefore, immobilization does not lead to any major

TABLE 1: Photophysical Propierties of Gramicidin(Emission Maxima, Fluorescence Lifetimes τi, NormalizedAmplitudes ri, Amplitude-Weighted Lifetimes τj, andAverage Fluorescence Lifetimes ⟨τ⟩) Inserted in EyPC andEyPG Liposomes Suspended in Solution or Immobilized in aSol-Gel Matrix; T ) 25 °C

systemλmax

(nm) R1 τ1 (ns) R2 τ2 (ns) τj (ns) ⟨τ⟩ �2

EyPC 332 0.78 1.1 0.22 3.2 1.5 2.0 0.87EyPC/sol-gel 332 0.75 1.1 0.25 3.3 1.6 2.2 0.87EyPG 328 0.79 0.7 0.21 2.0 1.0 1.3 0.87EyPG/sol-gel 328 0.80 0.7 0.20 2.1 1.0 1.3 0.98

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conformational change in the peptide. In contrast, a cleardecrease is observed for the entrapped zwitterionic system, inwhich the kQ,eff value dropped from 1.13 to 0.67 M-1ns-1. Thisreduction in the quenching efficiency could be due to either aconformational change in the entrapped peptide or to a slowerdiffusion of the quencher through the sol-gel pores. The firstpossibility is not likely, taking into account that lifetime andfluorescence spectra of gramicidin incorporated in EyPC werenot practically affected upon immobilization. The secondalternative is most likely to be correct because of the possibleexistence of electrostatic interactions between the polar headsof the zwitterionic phospholipids and the negatively chargedpores of the host matrix, which have been reported recently byour group for immobilized liposomes in the absence ofgramicidin.17 Such interactions would make more difficult thediffusion of acrylamide through the pores, reducing its acces-sibility to the peptide.

Effect of Sol-Gel on the Physical State of the GramicidinLipid Systems. To investigate the effects of sol-gel im-mobilization on the physical state of the lipid systems containinggramicidin, we used the reporter molecule Laurdan previouslyincorporated into the membrane. Laurdan is a polarity-sensitivefluorescent probe localized at the level of the glycerol backboneof the lipid bilayer whose fluorescent spectra depend stronglyon the lipid packing.29,30,42,43 Upon excitation, the dipole momentof Laurdan increases noticeably and water molecules in thevicinity of the probe reorient around this new dipole. When themembrane is in a fluid phase, the reorientation rate is fasterthan the emission process and, consequently, a red-shift isobserved in the emission spectrum of Laurdan. However, whenthe bilayer packing increases part of the water molecules isexcluded from the bilayer and the dipolar relaxation of theremaining water molecules is slower, leading to a fluorescentspectrum which is significantly less shifted to the red. The insertsof Figure 3 show the fluorescence emission spectra of Laurdan

recorded in LUVs of EyPC and EyPG, in the presence andabsence of gramicidin, freely suspended in buffer and im-mobilized in a sol-gel matrix. For the negatively charged lipidsystems, emission spectra are typical of a pure fluid phase andremain practically unmodified with encapsulation. This resultindicates that the physical properties of the anionic bilayer, withor without gramicidin, are not altered within the sol-gel pore.This is not the case for the immobilized zwitterionic systems,whose emission spectra are slightly blue-shifted as comparedto solution. This spectral behavior, indicative of a morehydrophobic and packed environment surrounding Laurdan, isin agreement with that obtained for immobilized DMPC17 andcan be caused by the electrostatic interactions establishedbetween the outer layer of the zwitterionic bilayer and the silanolgroups placed at the porous surface of the host matrix. Suchinteractions do not depend on the presence of gramicidin, assimilar results were obtained when peptide was incorporatedinto the membrane.

More information regarding the physical state of the im-mobilized systems can be inferred by measurement of thegeneralized polarization (GPex) of Laurdan. A high GPex valueis usually associated with a high bilayer packing and a lowpolarity, whereas a low GPex value is the opposite. Fulldiscussions of the use and mathematical significance of thisparameter have been published elsewhere.29,30 Figure 3 showsthe plot of GPex versus excitation wavelength for LUVs ofEyPG, gramicidin-EyPG, EyPC, and gramicidin-EyPC freelysuspended in buffer and immobilized in a sol-gel matrix. Theresults show that GPex values for the immobilized anionicsystems are very close to that in solution and exhibit what isconsidered a regular behavior; that is, the GPex spectrum exhibitsa clear tendency to decrease with the increase of excitationwavelength (as corresponds to a fluid phase). However, this wasnot the case for the immobilized zwitterionic liposomes.Regardless of the fact that GPex decreases with the increase ofexcitation wavelength, the values are higher than the ones foundin solution, probably due to hindering permeation of water intothe bilayer. This result supports the conclusions derived fromthe Laurdan emission spectra and confirms that, as a conse-quence of the liposome-silica interactions, the immobilizedEyPC and gramicidin-EyPC membranes behave as if they werein a pure fluid phase but remaining in a more rigid state.

Ionic Channel Activity. Activity of gramicidin reconstitutedin liposomes entrapped in sol-gel matrices was determined bya method based on the measurement of an ion flux through thepeptide. This is an alternative to the method used by Brennan’sgroup,24 where the activity of the ion channel is indirectlyevaluated through the establishment of a membrane potential.As is described in Materials and Methods, the activity assay isbased on the fluorescence quenching of the hydrophilic fluo-rophore PTSA, previously entrapped in the aqueous phase ofliposomes, by an externally added cation quencher as Cs+.44

When the cation permeates into the vesicles containing the ionicchannel, collisional quenching occurs between the ion and thetrapped PTSA. For the lipid systems in solution, this processwas very fast, and fluorescence intensity of PTSA decreased ina few seconds after addition of increasing amounts of Cs+.Experimental data were analyzed in terms of eq 2. TheStern-Volmer plots were linear (Figure 4), and the KSV valuesextracted from the slopes were 1.10 ( 0.08 and 1.71 ( 0.01for the zwitterionic and the anionic system, respectively. Thisresult confirms that the channel conformation of the peptide ismaintained in both types of membranes and suggests that thechannel activity is much larger in the anionic membrane than

Figure 2. Stern-Volmer plots for acrylamide quenching of gramicidinfluorescence in EyPC and EyPG liposomes freely suspended in buffer(+) and entrapped in a sol-gel matrix (O).

TABLE 2: Acrylamide Quenching Constants (KSV,eff andkQ,eff) of Gramicidin Inserted in EyPC and EyPG LiposomesSuspended in Solution or Immobilized in a Sol-Gel Matrix;T ) 25 °C

system KSV,eff (M-1) kQ,eff (M-1ns-1)a

EyPC 2.26 ( 0.02 1.13 ( 0.05EyPC/sol-gel 1.48 ( 0.09 0.67 ( 0.08EyPG 1.16 ( 0.02 0.89 ( 0.09EyPG/sol-gel 1.28 ( 0.07 0.98 ( 0.12

a Determined from kQ,eff ) KSV,eff/⟨τ⟩.

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in the zwitterionic one. This enhancement of conductance wouldbe in agreement with the results obtained by other groups41,45

and has been attributed to the strong electrostatic interactionsbetween the Cs+ and the negatively charged membrane surfacewhich increase the local concentration of the cation near thechannel entrance.

For the immobilized systems, the diffusion constraintsimposed by the dimensions of the monoliths and the restrictionto the free diffusion of Cs+ through the sol-gel matrix requireda longer time to complete the PTSA quenching process ascompared to the bulk solution. To evaluate this time, themonoliths were placed in vials containing 250 mM CsCl andchanges in the fluorescence intensity were measured at differenttimes. The inserts of Figure 4 show that the fluorescence ofPTSA trapped in the anionic system decreases in a similar wayto that observed for the zwitterionic one, to reach a constantvalue after 90-100 min. This time was selected as theincubation time for the rest of activity assays. Results indicatethat gramicidin incorporated in EyPG and EyPC liposomesretains its activity upon sol-gel encapsulation. To compare therelative activity of the channel before and after immobilization,the efficiency of the quenching process was evaluated afteraddition of increasing amounts of Cs+ (Figure 4). As in solution,the Stern-Volmer plots were linearly dependent on the quencherconcentration and KSV was directly extracted from the slope ofthe line. For the anionic system, KSV decreased from 1.71 (0.01 to 1.23 ( 0.04 upon immobilization, a value very close to

that obtained for the zwitterionic system freely suspended inbuffer. This result could be attributed to the lost or weakeningof the interactions, reported in solution, between the EyPGmembrane and the positively charged Cs+ because of thepresence of negative charges in the host matrix. For theimmobilized zwitterionic system, KSV was 1.03 ( 0.02, a valuevery similar to that recovered before entrapment, indicating thatthe ionic channel properties of the immobilized gramicidin arepreserved even though the zwitterionic polar heads interact withporous surface of the host matrix.

Conclusions

The goal of this study has been to analyze the effect of theinteractions of the phospholipid polar headgroups with thesilica-gel matrix on the biophysical and functional propertiesof gramicidin. With this purpose, we have characterized thedifferences in the conformation, solvent accessibility, andactivity of the peptide but in addition we have determined thestate of the lipid bilayer, a point that has not been done beforefor membranes entrapped in sol-gel matrices containingmembrane proteins. Before immobilization, significant differ-ences were found in the photophysical properties of gramicidinwhen inserted either in the anionic or in the zwitterionic bilayer,suggesting that peptide adopts a different conformation in bothlipid systems. These two conformations should be compatiblewith the gramicidin channel form, as ion flux across themembrane was demonstrated in both cases. Immobilization ofthe anionic system in the sol-gel matrix did not affect eitherthe physical properties of the bilayer or the channel activityof the gramicidin. In contrast, for the zwitterionic system theelectrostatic interactions established between the lipid polarheads and the sol-gel walls altered the membrane packing and,consequently, the physical properties of the membrane but didnot modify neither the conformation of the peptide nor its ion-channel activity. These results suggest that membrane proteinsreconstituted in zwitterionic bilayers and immobilized in sol-gelmaterials would preserve their function provided that suchfunction does not depend on the structural integrity and fluidityof the bilayer. In this case, anionic liposomes instead ofzwitterionic liposomes should be employed to minimize theinteraction with the negative-charged silica surface of the porousmatrix. However, if more complex membrane proteins withextracellular hydrophilic domains are used, it is possible thatthese domains could also interact with the host matrix aszwitterionic polar head groups do, possibly affecting theirfunction. In such cases, it should be advisible the incorporationduring the immobilization process of additive agents such as

Figure 3. Excitation generalized polarization (GPex) and normalized emission spectra (insert) of Laurdan in EyPC and EyPG liposomes recordedunder different conditions: a) freely suspended in buffer, in absence (s) and presence (O) of gramicidin; and b) entrapped in a sol-gel matrix, inabsence (---) and presence (•) of gramicidin.

Figure 4. Stern-Volmer plots for Cs+ quenching of PTSA fluores-cence. PTSA was encapsulated in gramicidin-EyPC and gramicidin-EyPG liposomes freely suspended in buffer (+) and entrapped in asol-gel matrix (O). (Inserts) Decrease with time of the fluorescenceintensity of PTSA in sol-gel immobilized gramicidin-EyPC andgramicidin-EyPG liposomes upon addition of 250 mM CsCl.

Characterization of an Ion Channel Peptide J. Phys. Chem. B, Vol. 113, No. 21, 2009 7539

polyethylenglycol, which serves as a protecting environmentfor the liposomes to avoid their interaction with sol-gel porouswalls.16,46 Further work is currently under progress in ourlaboratory to corroborate this issue, which includes the im-mobilization of ion channels and membrane enzymes of highermass and biological relevance.

Acknowledgment. The authors thank the Spanish Ministeriode Ciencia e Innovacion (MICINN) for grants MAT2005-01004and MAT2008-05670. R. Esquembre acknowledges the supportof a predoctoral fellowship from MEC.

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