Wilfrid Laurier University Scholars Commons @ Laurier Chemistry Faculty Publications Chemistry 2008 Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes Masoud Jelokhani-Niaraki Wilfrid Laurier University, [email protected] Robert S. Hodges University of Colorado -- Denver Joseph E. Meissner Wilfrid Laurier University Una E. Hassenstein Wilfrid Laurier University Laura Wheaton Wilfrid Laurier University Follow this and additional works at: https://scholars.wlu.ca/chem_faculty Recommended Citation Jelokhani-Niaraki, Masoud; Hodges, Robert S.; Meissner, Joseph E.; Hassenstein, Una E.; and Wheaton, Laura, "Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes" (2008). Chemistry Faculty Publications. 6. https://scholars.wlu.ca/chem_faculty/6 This Article is brought to you for free and open access by the Chemistry at Scholars Commons @ Laurier. It has been accepted for inclusion in Chemistry Faculty Publications by an authorized administrator of Scholars Commons @ Laurier. For more information, please contact [email protected]. 3306 Biophysical Journal Volume 95 October 2008 3306–3321 Interaction of Gramicidin S and its Aromatic Amino-Acid Analog with Phospholipid Membranes Masoud Jelokhani-Niaraki,* Robert S. Hodges,y Joseph E. Meissner,* Una E. Hassenstein,* and Laura Wheaton* *Department of Chemistry, Wilfrid Laurier University, Waterloo, Ontario, Canada; and yDepartment of Biochemistry and Molecular Genetics, University of Colorado Denver, School of Medicine, Anschutz Medical Campus, Aurora, Colorado ABSTRACT To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological mem- branes, a series of five decameric cyclic cationic b-sheet-b-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar [ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 $ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis. INTRODUCTION Interaction of antimicrobial peptides with cell membranes has tive bacteria as well as fungi, but is also highly hemolytic been considered as a fundamental, if not the only, step in the against mammalian erythrocytes (12). The physicochemical mechanism of their antimicrobial activity (1,2). It has been properties, synthetic procedures, and biological activity of GS known that helical cationic peptide antibiotics interact with and tyrocidines, as well as hundreds of their different an- cell membranes of both prokaryotic and eukaryotic cells, alter alogs, have been investigated in detail (6,7,13). or destroy the cell membrane integrity, and cause lysis leading The sequences of GS and its five analogs are shown in to the cell death (3,4). In comparison to the linear helix- Table 1. The peptides share two type II9 b-turns composed of forming cationic antimicrobial peptides, the mechanism of four amino acids (-Leu-D-Xxx-Pro-Val-, where Xxx is an interaction of cyclic cationic antimicrobial peptides (CCAPs) aromatic amino acid) on the two ends of an antiparallel with biological membranes is much less understood (5). To b-sheet, stabilized by four backbone intramolecular hydrogen investigate the mechanism of interaction of small CCAPs with bonds. In addition to sharing the b-sheet-b-turn conforma- biological model membranes, we have designed and synthe- tion, the peptides of Table 1 are also amphipathic. The hy- sized a series of amphipathic decameric b-sheet-b-turn cyclic drophobic residues (Val and Leu) in these peptide molecules peptides with different combinations of aromatic D-amino- are located on one face and Lys (Orn in the case of GS) res- acid (phenylalanine, tyrosine, and tryptophan) substitutions. idues are located on the opposite face. A typical structure and The prototypic models for the peptides of this study are the orientation of the amino-acid residues of the peptides of Table naturally found decameric CCAPs, gramicidin S (GS), and 1 are depicted in Fig. 1, a representation of the three-dimen- tyrocidines of the same bacterial origin (6,7). sional structure of GS supported by molecular modeling. Gramicidin S, the principal prototype for the peptides In this study, we have utilized the biophysical techniques, of this study, is a CCAP from the Gram-positive bacteria circular dichroism (CD), fluorescence spectroscopy, and Bacillus brevis that has been widely utilized as a topical isothermal titration calorimetry (ITC), to explore the con- antibiotic (8). The backbone structure of GS is comprised of a formation and modes of interaction of aromatic b-sheet-b- double-stranded antiparallel b-sheet connected by a pair turn amphipathic CCAPs with model biological membranes of type II9 b-turns on both ends (Fig. 1) (9–11). GS is active and suggested a model for the mechanism of interaction of against a broad range of both Gram-positive and Gram-nega- these peptides with lipid membranes. MATERIALS AND METHODS Submitted May 14, 2008, and accepted for publication June 30, 2008. Chemicals Address reprint requests to Masoud Jelokhani-Niaraki, Tel.: 519-884-1970, Ext. 2284; E-mail: [email protected]. Gramicidin S was purchased from Sigma (St. Louis, MO) and further purified Editor: Paul H. Axelsen. by RP-HPLC. All other peptides were synthesized by solid-phase procedures Ó 2008 by the Biophysical Society 0006-3495/08/10/3306/16 $2.00 doi: 10.1529/biophysj.108.137471 Cyclic Peptide-Membrane Interaction 3307 assay (12,14). The reported values for both antimicrobial and hemolytic assays were determined after 24 h at 37°C. Large unilamellar vesicles for spectroscopic and ITC experiments Chloroform solutions of the lipids POPC and POPE/POPG (7/3 molar ratio) were dried, under a mild nitrogen flush, in a round-bottomed flask to form a thin film. The film was further dried overnight under reduced pressure. The dried lipid film was then hydrated with buffer at room temperature. The re- FIGURE 1 Molecular model for the amphipathic b-sheet-b-turn confor- sulted multilamellar vesicles were then freeze-thawed a few times and ex- mation of GS. truded through a 100-nm filter in a LiposoFast apparatus (Avestin, Ottawa, Canada) (15). The prepared large unilamellar vesicles (average diameter of ;80 nm) were stable in dark for several days at 4°C. using tert-butyloxycarbonyl (Boc) chemistry. N-[(1H-benzotriazol-1yl) Two phospholipid vesicle systems were utilized for this study. The first (dimethylamino)methylene]-n-methylmethanaminium hexafluorophosphate system contained POPC lipid bilayers, which can be considered as a model n-oxide (HBTU) and 1-hydroxybenzotriazole (HOBT) were used to activate for the mammalian blood cell membranes. The second system contained C-terminus of the amino acids. Lys side chains were protected by formyl POPE and POPG phospholipids ([phosphatidylethanolamine (PE)]/[phos- groups throughout the syntheses. After cleavage from resin and purification phatidylglycerol (PG)] ¼ 7/3) to model the negatively-charged bacterial by RP-HPLC, linear peptides were cyclized in a head-to-tail manner (Pro membranes. Similar to biological membranes, the two lipid bilayer systems at the C-terminus) utilizing benzotriazole-1-yl-oxy-tris-(dimethylamino)- used for the biophysical measurements in this study were in the liquid phosphonium hexafluorophosphate (BOP), 1-hydroxybenzotriazole, and n, crystalline state. n-diisopropylethylamine in n,n-dimethylformamide, and Lys residues of the cyclized peptides were then deprotected in dilute methanolic HCl at 40°C (12,14). Peptides were purified and analyzed by RP-HPLC, and their final purity was Instrumentation further confirmed by electrospray mass spectrometry (Table 1). Concentration of the pure peptides in aqueous stock solutions was determined by amino-acid Peptides were analyzed on a Waters 600E HPLC system (Waters, Franklin, analysis (65% error). Phospholipids, 1-palmitoyl-2-oleoyol-sn-glycero-3-phos- MA) using a Waters C4 Symmetry (150 3 4.6 mm I.D., 5 mm particle size, phocholine (POPC), 1-palmitoyl-2-oleoyol-sn-glycero-3-phosphoethanolamine 30 nm pore size) at 25°C. CD spectra were measured on an Aviv 215 (POPE), and 1-palmitoyl-2-oleoyol-sn-glycero-3-[phosphor-rac-1-glycerol] spectropolarimeter (Aviv Biomedical, Lakewood, NJ). Ellipticities are (sodium salt) (POPG) were from Avanti Polar Lipids (Alabaster, AL). Stock reported as mean residue ellipticity, [u]. Spectra were