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Functional Modi¢Cations of Alamethicin Ion Channels by Substitution of Glutamine 7, Glycine 11 and Proline 14

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Functional Modi¢Cations of Alamethicin Ion Channels by Substitution of Glutamine 7, Glycine 11 and Proline 14 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1373 (1998) 137^146 Functional modi¢cations of alamethicin ion channels by substitution of glutamine 7, glycine 11 and proline 14 Christine Kaduk, Margitta Dathe *, Michael Bienert Forschungsinstitut fu«r Molekulare Pharmakologie, Alfred-Kowalke-Str. 4, 10315 Berlin, Germany Received 4 February 1998; revised 9 June 1998; accepted 11 June 1998 Abstract Alamethicin is a 20 amino acid, potentially helical peptaibol which forms voltage-dependent ion channels in bilayer systems. Two aspects of alamethicin structure have been suggested to be of particular functional significance for stabilization of alamethicin channels. (i) Proline 14 inducing a helix kink is together with glycine at position 11 responsible for an appropriate orientation of the molecules in the conducting associates. (ii) Glutamine 7 lining the channel interior is assumed to stabilize the channel structure by forming inter-helix hydrogen bonds. The functional importance of these residues was probed in macroscopic and single-channel experiments with alamethicin analogs containing polar, side chain bearing residues at position 11 (glutamine, asparagine) or at position 14 (glutamine). In order to investigate the crucial role of glutamine 7 for the stabilization of channel aggregates, this residue was substituted by alanine. The conformation of the lipid bound peptides was determined by circular dichroism spectroscopy. The results show that glutamine 7, glycine 11 and proline 14 are not essential for channel formation but substitution of any residue reduced the number of conductance levels and significantly reduced their lifetimes. Channel stabilization by the introduction of residues with potential hydrogen bonding capacity at positions 11 and 14 was not observed. Differences in the conformation of the lipid bound peptides, their orientation in the bilayer and their affinity for the lipid membrane appear thus to contribute to the modulation of functional properties. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Alamethicin analog; Voltage-dependent ion channel; Conductance; Lipid bilayer; Peptaibol; Conformation 1. Introduction interaction with lipid bilayers, biological properties and ion channel-forming behavior have been inten- Alamethicin is a member of the peptaibol family sively investigated (for review see [2,3]). While the of channel-forming peptides. The linear 20 amino alamethicin chain is very £exible in aqueous solu- acid containing peptide which was originally iso- tion, the tendency of Aib to restrict the possible lated from the fungus Trichoderma viride [1] is char- conformers of peptides is con¢rmed in the case of acterized by a high proportion of K-aminoisobutyric alamethicin by its predominantly helical crystal acid (Aib), an acetylated N terminus and an K-ami- structure [4] and its largely helical conformation in no alcohol at the C terminus. Its conformation, methanol, in the associated state and when bound to lipids [5^8]. The N-terminal helix is highly hydro- phobic, while the C terminus is more polar and * Corresponding author. Fax: +30 (51) 551206; bent away from the helix axis by the proline residue E-mail: [email protected] at position 14. Thus the peptide appears to be 0005-2736 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S0005-2736(98)00100-X BBAMEM 77419 1-8-98 138 C. Kaduk et al. / Biochimica et Biophysica Acta 1373 (1998) 137^146 ideally suited for the N-terminal insertion into the The Gln7-Ala7 substitution abolished channel-form- membrane interior. ing activity which was, however, restored in the Ser The voltage-dependent formation of alamethicin analog and quite well developed in the Asn contain- ion channels has been described by di¡erent models ing sequence, thus supporting the concept of a stabi- [9]. Evidence for the `barrel-stave' model, which ex- lizing role for a polar residue at this particular posi- plains the conductivity inducing property of the pep- tion. taibol by the formation of water ¢lled, transmem- Our study tested the signi¢cance of Pro-14, Gly-11 brane bundles of helical monomers, derives from and Glu-7 using analogs derived from the native ala- the interpretation of single-channel investigations methicin sequence. In order to con¢rm the crucial [10,11]. Association of up to 12 monomers has role of Gln-7 for the stabilization of channels the been suggested to determine the di¡erent conduc- residue was substituted by Ala. The importance of tance levels, and the asymmetry of the electrophysio- the conformational £exibility in the central chain re- logical properties point to a parallel orientation of gion was probed with alamethicin analogs containing the monomers in the conducting states. side chain bearing residues at position 11 (Gln, Asn). Two aspects of the alamethicin conformation have Additionally, the substitution of Gly by polar resi- been found to be of particular functional signi¢- dues with amide side chain functionality and di¡er- cance: (i) proline (Pro) at position 14 inducing a ent lengths was directed towards the question as to helix kink and (ii) glutamine (Gln) at position 7 lin- whether steric hindrance could contribute to channel ing the channel interior. Remarkably, these residues stabilization via an intermolecular hydrogen-bond are highly conserved in many channel-forming pep- annulus at this position. Furthermore, the tendency tides [2]. The Pro induced break in the hydrogen of a polar residue with potential hydrogen-bonding bonding pattern results in the funnel shaped appear- capacity to contribute to channel stabilization at po- ance of the channel. The formation of the `mouth' sition 14 was studied by substitution of Pro-14 by structure at the C terminus is supported by the con- Gln. formationally £exible glycine 11 (Gly) [12]. Both the The functional properties of the alamethicin ana- lack of hydrogen bond constraints between Pro-14 logs were compared in both macroscopic and single- and Aib-10 and the absence of a restricting side channel conductance experiments after incorporation chain at position 11 have been suggested as contri- in a planar lipid bilayer and are discussed in relation buting to an optimal orientation of the monomers in to structural parameters and the conformation of the the gating alamethicin pores. The functional impor- lipid bound peptides as determined by circular di- tance of Pro-14 of alamethicin has been described in chroism. several investigations [13,14]. The residue was shown to be not essential for the voltage induced ion £ow through lipid bilayers. Any substitution at this par- 2. Materials and methods ticular position, however, was found to be connected with distinct conformational changes and substantial 2.1. Synthesis of peptides loss of single-channel activity re£ected by a reduced number and lifetime of conductance levels. The alamethicin peptides were synthesized by the Like Pro-14 and Gly-11, the polar side chain of solid phase technique using Fmoc protected amino Gln-7 is presumed to be directed towards the center acid £uorides which were prepared via (diethylami- of the channel lumen. Early investigations indicated no)sulfur tri£uoride [16]. The peptide assembly was that the residue might play a role in bundle stabiliza- carried out on a MPS synthesizer ACT 348 (Ad- tion by inter-helix hydrogen bonding between the vanced ChemTech, Louisville, KY, USA) as de- side chains [4]. Molle et al. [15] made an attempt to scribed [17]. After completion of the synthesis and verify the role of Gln-7 in channel formation by ala- acetylation of the N terminus, the peptides were re- methicin by investigating non-Aib-containing syn- leased from the resin by treatment with 2% triisopro- thetic analogs with substitution of Gln at position pylsilane and 5% water in 50% tri£uoroacetic acid 7 by alanine (Ala), serine (Ser) or asparagine (Asn). (TFA)/dichloromethane (DCM) for 45 min to give BBAMEM 77419 1-8-98 C. Kaduk et al. / Biochimica et Biophysica Acta 1373 (1998) 137^146 139 Table 1 Amino acid sequences of alamethicin F30 (Alam-P14) and the alamethicin analogs 1 7 11 14 20 Alam-P14: Ac-Aib-Pro-Aib-Ala-Aib-Ala Gln-Aib-Val-Aib- Gly-Leu-Aib- Pro-Val-Aib-Aib-Glu-Gln- Pheol Alam-Q14: Ac-Aib-Pro-Aib-Ala-Aib-Ala- Gln-Aib-Val-Aib- Gly-Leu-Aib- Gln-Val-Aib-Aib-Glu-Gln- Pheol Alam-A7: Ac-Aib-Pro-Aib-Ala-Aib-Ala Ala-Aib-Val-Aib- Gly-Leu-Aib- Pro-Val-Aib-Aib-Glu-Gln- Pheol Alam-Q11: Ac-Aib-Pro-Aib-Ala-Aib-Ala Gln-Aib-Val-Aib- Gln-Leu-Aib- Pro-Val-Aib-Aib-Glu-Gln- Pheol Alam-N11: Ac-Aib-Pro-Aib-Ala-Aib-Ala Gln-Aib-Val-Aib- Asn-Leu-Aib- Pro-Val-Aib-Aib-Glu-Gln- Pheol the N-acetylated free peptide. The peptides were to the relation [3]222 = 330 300 [K]+2340 ([K] = puri¢ed by reverse phase HPLC and characterized amount of helix) [18]. The error was 5% helicity. by electrospray ionization mass spectrometry (ESI- MS). 2.3. Macroscopic and single-channel conductance experiments 2.2. Circular dichroism experiments Macroscopic and single-channel conductance ex- 2.2.1. Vesicle preparation periments were performed at planar lipid bilayers Small unilamellar vesicles (SUVs) for CD measure- formed across either a 175 Wm or 125 Wm diameter ments were prepared by suspending and vortexing hole in a 25 Wm thick Te£on ¢lm (PTFE; Goodfel- the dried 1-palmitoyl-2-oleoylphosphadidylcholine low, Cambridge, UK). The Te£on ¢lm was sand- (POPC) (Avanti Polar Lipids, Alabaster, AL, USA) wiched between two glass half-cells and pretreated in bu¡er (10 mM Tris, 154 mM NaF, 0.1 mM with hexane/hexadecane (40:1 v/v) (Fluka, spectros- EDTA, pH 7.4) to give ¢nal lipid concentrations copic grade).
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