Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranesIntroductionMaterials and methodsMaterialsPreparation of liposomesNBD-nystatin partitioning experimentsAbsorption and steady state fluorescence measurementsTime-resolved fluorescence measurementsData analysisResultsNBD-nystatin binding to the lipid vesiclesHetero-FRET measurements in the membraneFluorescence self-quenching and homo-FRET measurements between lipid-bound NBD-NysDiscussionSolution and membrane-bound conformations of the polyene antibiotic nystatinDetermination of the oligomeric state of the antibiotic in membranes by energy migrationConclusionsAcknowledgementsDetermination of energy transfer efficiencies for membrane-bound NBD-NysReferencesConformation and self-assembly of a nystatin nitrobenzoxadiazolederivative in lipid membranesLiana Silvaa, Ana Coutinhoa,b, Alexander Fedorova, Manuel Prietoa,*aCentro de Quı´mica-Fı´sica Molecular, Complexo Interdisciplinar, Instituto Superior Te´cnico, Av. Rovisco Pais, P-1049-001 Lisbon, PortugalbDepartamento de Quı´mica e Bioquı´mica, Bloco C8, Faculdade de Cieˆncias, Universidade de Lisboa, R. Ernesto de Vasconcelos,P-1749-016 Lisbon, PortugalReceived 21 May 2003; received in revised form 9 September 2003; accepted 11 September 2003AbstractNystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the presentstudy, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free andergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). Themean tetraene to NBD separating distance determined from fluorescence energy transfer measurements increased from 18 to 25.6 A˚uponantibiotic binding to the lipid vesicles, indicating that the monomeric labelled antibiotic adopts a more extended conformation in its lipid-bound state than in aqueous solution. The oligomeric state of membrane-bound NBD-Nys was also studied by resonance energy homotransferbetween the NBD fluorophores. The decrease measured in its steady state fluorescence anisotropy upon increasing the surface concentration ofthe NBD-Nys is shown to be consistent with a random distribution of molecules on the surface of the liposomes. This data contradicts the sharpincrease measured for nystatin mean fluorescence lifetime in the presence of 10 mol% ergosterol-containing POPC LUV within the sameantibiotic concentration range and which is known to report nystatin oligomerization in the lipid vesicles. Therefore, we conclude that theamine group of nystatin is an essential requisite for the supramolecular organization/pore formation of this antibiotic.D 2003 Elsevier B.V. All rights reserved.Keywords: Antifungal; Polyene macrolide antibiotic; Fluorescence; Nystatin; Energy migration1. IntroductionNystatin (Nys) and amphotericin B (AmB) are antifungalantibiotics that permeabilize a wide spectrum of eukaryoticcells, including fungal and mammalian cells, and syntheticlipid vesicles [1]. Membrane sterols are believed to play arole in the antibiotic’s activity and it has been proposed thatthe selective toxicity of these polyene antibiotics for fungiresults from their capacity to bind more strongly to ergos-terol, the main fungal sterol, than to cholesterol, the mainsterol present in mammalian cells [1]. Prevailing models forthe mechanism of action of Nys and AmB postulate theformation of transmembrane channel s with size-discriminat-ing properties in the plasma membrane of the antibiotic-sensitive organisms [2]. Recently, Gruszecki et al. [3]detected the formation in monolayers of cylindrical struc-tures of AmB with an internal diameter close to 0.6 nm byscanning force microscopy. However, there is no consensuson the pathway of induction of membrane damage by theseantibiotics or on the role played by sterols in this process [4].Several authors have proposed a sequence of three events:binding of the antibiotic monomer to the membrane surface,self-assembly to a prepor e oligomer, and insertion in the lipidbilayer generating a functional po re that mediates passiveflux of mol ecules across the membrane [5–7]. Cohen [7,8]further suggested that the initially formed nonaqueous chan-nels subsequently interacted with the sterols in the mem-brane to form aqueous channels, having an enlargeddiameter. Alternatively, Bolard et al. [9] proposed thatsoluble monomeric AmB is very active towards ergosterol-containing membranes , where classic barrel-stave-type com-0005-2736/$ - see front matter D 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamem.2003.09.004Abbreviations: AmB, amphotericin B; LUV, large unillamelar vesicles;NBD-Nys, (6-[N-(4-nitro-2,1,3-benzoxadiazolyl)-aminohexanoyl]nystatin;Nys, nystatin; P10C, 10 mol% cholesterol-containing POPC LUV; P10E,10 mol% ergosterol-containing POPC LUV; POPC, 1-palmitoyl-2-oleoyl-1-sn-glicero-3-phosphocholine* Corresponding author. Tel.: +351-21-8419219; fax: +351-21-8464455.E-mail address: [email protected] (M. Prieto).www.bba-direct.comBiochimica et Biophysica Acta 1617 (2003) 69 – 79plexes with this sterol are formed, but a soluble self-associ-ated and sterol-free oligomer is required for the permeabili-zation of cholesterol-containing membranes. On the otherhand, other studies have demonstrated the ability of AmB toform channels in sterol-free membranes under specific con-ditions like when using high antibiotic concentrations [10]and in osmotically stressed liposomes [11,12].Previous studies from our laboratory have shown thatNys mean fluorescence lifetime is a reporter parameter of itsaggregation state in gel-phase DPPC small unilamellarvesicles (SUV) and large unilamell ar vesicles (LUV)[13,14] and in ergosterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) LUV [15]. Surpris-ingly, the fluorescence emission decay kinetics of Nys wasfound to remain essentially invariant in the presence ofcholesterol-containing liposomes [15], although indepen-dent data from activity assays suggested t hat aqueouschannels were also formed in these lipid vesicles (Coutinhoand Prieto, unpublished work). We proposed that Nyschannel architecture must be sterol-dependent, its structurebeing looser and less stable in cholesterol-containing lipidmembranes than in ergosterol-containing lipid membranes.This hypothesis stems