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Biophysical and Functional Characterization of an Ion Channel Peptide Confined in a Sol-Gel Matrix Rocı´o Esquembre, Jose´ Antonio Poveda, and C. Reyes Mateo* Instituto de Biologı´a Molecular y Celular. UniVersidad Miguel Herna´ndez 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 the bilayer fluidity, which can limit the use of these systems for practical applications. Recently, we have reported that electrostatic interactions between phospholipids polar heads and the negative-charged silica surface of the porous matrix should be the cause of such behavior. In the present work, we analyze the effect of these interactions on the biophysical and functional properties of the ion-channel peptide gramicidin, entrapped in a sol-gel matrix, to get more insight on the ability of these inorganic materials to immobilize ion channels and other membrane-bound proteins. Gramicidin was reconstituted in anionic and zwitterionic liposomes and the effects of sol-gel immobilization on the biophysical properties of gramicidin were determined from changes in the photophysical properties of its tryptophan residues. In addition, the physical state of the immobilized lipid membrane containing gramicidin was analyzed by measuring the spectral shift of the fluorescent probe Laurdan. Finally, the ion-channel activity of the peptide was monitored upon sol-gel immobilization through a fluorescence quenching assay using the fluorescent dye pyrene-1,3,6,8-tetrasulfonic acid (PTSA). Results show that the channel properties of the immobilized gramicidin are preserved in both zwitterionic and anionic liposomes, even though the zwitterionic polar heads interact with the porous surface of the host matrix. Introduction Ion channels are membrane proteins involved in the maintenance of the appropriate ion balance across biological membranes, connecting the inside of the cell to its outside in a selective fashion. They are natural nanotubes, which serve as key elements in signaling and sensing pathways and thus govern an enormous range of biological functions important in health and disease.1 The ability to immobilize these proteins in inorganic matrices represents a significant step forward in developing a new generation of biologically active materials with potential applications in areas such as high-throughput drug screening and new generation of biosensors.2 The major limitation in the development of these new materials involves the difficulty of finding immobilization techniques able to retain the physical properties of the lipid bilayer where the ion-channel activity is included. Because of their dynamic and self-assembled nature, fixation of lipid membranes to solid surfaces easily disrupts the hydrophobic interactions that create lipid bilayers and modifies the natural dynamic motions of the membrane and its thermotropic properties, producing unstable immobilized structures and, in some cases, the rupture of the bilayer. Different strategies have been developed to overcome these difficulties.3 Among them, use of sol-gel materials seems to be an interesting alternative to immobilize lipid membranes vesicles (liposomes) and membrane-bound proteins in solid matrices without the need for tethering the lipids to a surface.4,5 This sol-gel methodology has been extensively used to immobilize soluble proteins showing that the majority of them can be encapsulated with retention of their native structure and functionality and an enhanced stability.6-10 Bioencapsulation within these materials is currently obtained from the hydrolysis of alkoxide precursors * To whom correspondence should be addressed. Tel.: +34 966 658 469. Fax: +34 966 658 758. E-mail:
[email protected].
(usually tetraethyl orthosilicate, TEOS, or tetrametyl orthosilicate, TMOS) resulting in a colloidal sol solution. Subsequently, a buffered aqueous solution containing the biomolecule of interest is added to the sol producing a polycondensation reaction that leads to the formation of a transparent highly porous gel that 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 heavymetal ion and pH sensing, respectively. A detailed characterization of the physical properties and stability of the immobilized lipid systems was carried out later by the Brennan group.13 They incorporated liposomes composed of dipalmitoyl phosphatidylcholine (DPPC) in a TEOS sol-gel matrix and observed that the lipid bilayer did not exhibit phase transition suggesting that encapsulation resulted in rupture of these structures. This behavior was attributed to interactions between the lipid and the ethanol resulting as a byproduct of the chemical reactions involved in the formation process of the silica matrix. A similar conclusion was reached by Halder et al.14 when encapsulating dimyristoyl phosphatidylcholine (DMPC) in TEOS glasses. To overcome these problems, use of either new precursors (other than TEOS and TMOS), which minimize the generated alcohol,13,15 or alternative alcohol free routes16,17 is required. Nevertheless, it has been reported that, for pure zwitterionic liposomes, (i.e., DMPC, DPPC) the lipid phase transition is affected although these routes are used, suggesting irreversible alterations of the bilayer fluidity that could prevent the use of these systems for practical applications.13,18 Recently, we have reported that electrostatic interactions between phospholipids’ polar heads and the negative-charged silica surface of the porous matrix seem to play a capital role for the preservation of the structural integrity of the immobilized bilayer.17 Here, we analyze the effect of such interactions on the biophysical and functional properties of the model ion-channel gramicidin to
10.1021/jp9019443 CCC: $40.75 2009 American Chemical Society Published on Web 05/04/2009
Characterization of an Ion Channel Peptide get more insight on the ability of these inorganic materials to immobilize ion channels and other membrane-bound proteins. Gramicidin is a pentadecapeptide antibiotic that inserts into the lipid bilayer forming prototypical ion channels specific for the transport of monovalent cations across membranes.19,20 This peptide has one of the most hydrophobic sequences known and is very sensitive to the environment in which it is placed, adopting a wide range of conformations, depending on the nature of solvent in which it is dissolved and on the incorporation method.19-21 In membranes, the most preferred conformations are the single-stranded helical dimer (the channel form) and the double-stranded interwined helix (the nonchannel form), which show different fluorescent properties.20 Recently, the elucidation of the X-ray crystal structure of the Streptomyces liVidans K+ channel (KcsA) in molecular detail22 has allowed to verify that the gramicidin channel form shares important structural features with real protein ion channels.21,23 Consequently, this peptide appears as a useful model for realistic determination of the effects of sol-gel immobilization on the biophysical and functional properties of membrane-bound proteins. Successful immobilization of the gramicidin channel form in sol-gel materials was reported for the first time by Brennan’s group using as silica precursor a diglycerilsilane derivative synthesized by themselves.24 However, when they tried to entrap this ionic channel using the traditional silica precursors TMOS or TEOS, results were unsuccessful, probably due to the abovementioned damaging effects of the alcohol generated during the hydrolysis process. Recently, in a preliminary work we managed to immobilize gramicidin in a TMOS sol-gel matrix using an alcohol-free route developed by Ferrer et al.,25 obtaining a system that was able to generate ion fluxes.18 In the present work, we have used the same methodology to immobilize gramicidin reconstituted in zwitterionic and anionic liposomes, and the effects of the phospholipids polar heads on the biophysical and functional properties of the immobilized peptide have been analyzed through steady-state and time-resolved fluorescence techniques. Fluorescence spectra, quenching experiments, and fluorescence lifetimes of the gramicidin tryptophans indicate that the biophysical properties of the reconstituted ionic channel are preserved in both zwitterionic and anionic liposomes upon sol-gel immobilization, but the lipid bilayer physical state, studied by the fluorescent probe Laurdan, is altered only in the case of zwitterionic phospholipids. Ionchannel activity of the immobilized gramicidin was monitored through a fluorescence quenching assay using the fluorescent dye pyrene-1,3,6,8-tetrasulfonic acid (PTSA). Results show that, contrary to that expected, the ionic channel activity is preserved in both zwitterionic and anionic liposomes, even though the zwitterionic polar heads seem to interact with the porous surface of the host matrix. Materials and Methods Chemicals. The precursor tetramethyl orthosilicate (TMOS) and the phospholipids phosphatidylglycerol (EyPG) and phosphatidylcholine (EyPC), both derived from egg yolk, were purchased from Sigma-Aldrich Chemical Co. (Milwauke, WI, USA). Gramicidin A′ and the fluorescent probes 2-dimethylamino-6-lauroylnaphtalene (Laurdan) and 1,3,6,8-pyrene tetrasulfonate (PTSA) were from Molecular Probes (Eugene, OR, USA) and used without further purification. Gramicidin A′, as purchased, is a mixture of gramicidins A, B, and C. Water was twice-distilled in all-glass apparatus and deionized using Milli-Q equipment (Millipore, Madrid, ES). Ethanol, methanol, and
J. Phys. Chem. B, Vol. 113, No. 21, 2009 7535 chloroform (spectroscopic grade) were from Merck. All other compounds were of analytical grade. Liposome Formation and Gramicidin Reconstitution. Chloroform/methanol solutions containing 3 mg of total phospholipid (EyPC or EyPG) were dried first by evaporation under a dry nitrogen gas stream and subsequently under vacuum for 3 h. Multilamellar vesicles (MLVs) were formed by resuspending the dried phospholipid in buffer (Tris-HCl 50 mM, NaCl 250 mM, pH 7.4) to a final concentration of 1 mM. The vesicle suspension was vortexed several times. Large unilamellar vesicles (LUVs) with a mean diameter of 90 nm were prepared from these MLVs by pressure extrusion through 0.1 µm polycarbonate filters (Nucleopore, Cambridge, MA). Gramicidin A’ was incorporated into MLVs as described previously.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 and after under vacuum, hydrated with 50 mM Tris-HCl buffer (pH 7.4, 250 mM NaCl), and vortexed to obtain MLVs and, subsequently, LUVs as described above. The sample was incubated overnight at 65 °C with continuous stirring to induce the channel-forming β-helical monomeric conformation.27,28 Labeling of Liposomes. A few microliters from a stock solution of the fluorescent probe Laurdan in ethanol was added to the LUVs’ suspension. Solutions were incubated for 30 min at 30 °C to facilitate the probe incorporation into the lipid bilayer. The ethanol final concentration was always less than 2%. The lipid-to-probe ratio, in molar terms, was 500:1. For samples to be used in the ion-channel activity assay, the fluorescent dye PTSA was encapsulated into the aqueous interior of LUVs formed as described above, except that the dried phospholipid was resuspended in a buffer containing PTSA 4.16 mM. Removal of the external fluorophore was accomplished by 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 pure silica matrices using an alcohol-free route developed by Ferrer et al.25 Silica sol stock solution was prepared by mixing 5.88 mL 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 deionized water and submitted to rotaevaporation for a weight loss of ∼0.6 g (i.e., 0.6 g are approximately the alcohol mass resulting from alkoxyde hydrolysis). The aqueous sol was mixed with 1 mL of a diluted buffered suspension of liposomes in a disposable cuvette of polymethylmethacrylate. Gelation occurs readily after mixing. Afterward, monoliths (9 mm × 9 mm × 17 mm) were wet aged in a Tris buffer (50 mM, pH 7.5, NaCl 250 mM) solution at 4 °C before use. Steady-State Fluorescence Measurements. Fluorescence measurements of gramicidin were performed in a SLM-8000C spectrofluorimeter (SLM Instruments Inc., Urbana, IL, USA). Emission spectra were excited at 290 nm with emission collected from 305 to 400 nm. The experimental samples (sol-gel monoliths and lipid suspensions) were placed in 10 × 10 mm cuvettes. Background intensities due to the sol-gel matrix and/ or liposomes were always taken into account and subtracted from the sample. Fluorescence emission spectra of Laurdan were obtained with a Cary Eclipse spectrofluorometer (Varian) interfaced with a Peltier cell. Emission spectra were recorded at 25 °C, fixing excitation wavelength at 350 nm. The spectral changes of the
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emission spectrum of the probe were also quantified by the socalled generalized polarization (GPex),29,30 which is defined as eq 1:
GPex )
I440 - I490 I440 + I490
(1)
where I440 and I490 are the fluorescence intensities recorded after subtraction of background intensity at the characteristic emission wavelengths of the gel phase (440 nm) and of the fluid phase (490 nm). GPex values were obtained in a range of excitation wavelengths (325-410 nm) at 25 °C. Time-Resolved Fluorescence Measurements. The decay of the total fluorescence intensity was recorded at 25 °C using a PTI model C-720 fluorescence lifetime instrument (Photon Technology International Inc., Lawrenceville, NJ) utilizing a proprietary stroboscopic detection technique.31 The system employs a PTI GL-330 pulsed nitrogen laser pumping a PTI GL-302 high-resolution dye laser. The dye laser output at 594 nm was frequency-doubled to 297 nm with a GL-303 frequency doubler coupled to an MP-1 sample compartment via fiber optics. The emission was observed at 90° relative to the excitation via an M-101 emission monochromator and a stroboscopic detector equipped with a Hamamatsu 1527 photomultiplier. The kinetic parameters of the impulse response fluorescence intensity decay i(t) ) ∑i Ri exp (- t/τi), lifetimes τi, and normalized amplitudes Ri, were recovered with the Felix 32 analysis package using a discrete one- to four-exponential fitting program. The amplitude-weighted lifetime proportional to quantum yield, jτ, and the average fluorescence lifetime, 〈τ〉, were calculated according to jτ ) ∑Riτi and 〈τ〉 ) (∑i Riτi2)/(∑i Riτi), respectively.32 The fits tabulated represent the minimum set of adjustable parameters that satisfy the usual statistical criteria, namely a reduced χ2 value of
