2D-NMR and ATR-FTIR Study of the Structure of a Cell-Selective Diastereomer of Melittin and Its Orientation in Phospholipids ² Michal Sharon, ‡ Ziv Oren, § Yechiel Shai, § and Jacob Anglister* ,‡ Department of Structural Biology and Department of Biological Chemistry, The Weizmann Institute of Science, RehoVot 76100, Israel ReceiVed May 28, 1999; ReVised Manuscript ReceiVed September 10, 1999 ABSTRACT: Melittin, a 26 residue, non-cell-selective cytolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V 5,8 ,I 17 ,K 21 -melittin, D-amino acids at positions V 5,8 ,I 17 ,K 21 ) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V 5,8 ,I 17 ,K 21 -melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]-V 5,8 ,I 17 ,K 21 -melittin binds strongly and destabilizes only negatively charged phospholipid vesicles, in contrast to native melittin, which binds strongly also zwitterionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D]-V 5,8 ,I 17 ,K 21 -melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were done with both melittin and [D]-V 5,8 ,I 17 ,K 21 -melittin. The structure of the diastereomer was characterized by NMR in water, as well as in three different membrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, methanol, and dodecylphosphocholine/phosphatidylglycerol (DPC/ DMPG) micelles. The NMR data revealed an amphipathic R-helix only in the C-terminal region of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V 5,8 ,I 17 ,K 21 -melittin are oriented parallel to the membrane surface. This study indicates the role of secondary structure formation in selective cytolytic activity of [D]-V 5,8 ,I 17 ,K 21 - melittin. While the N-terminal helical structure is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural element for binding and insertion into zwitterionic membranes and for hemolytic activity. Melittin, a 26-residue highly basic, amphipathic polypep- tide (GIGAVLKVLTTGLPALISWIKRKRQQ-CONH 2 ), is the major component of the honey bee (Apis mellifera) venom (1), and is one of the most studied membrane-seeking polypeptides (2). Melittin possesses various biological activi- ties on membranes of different cells (for a review, see refs 2 and 3), i.e., lysis of mammalian cells (4, 5), lysis of bacteria (6, 7), membrane fusion (8, 9), and activation of phospho- lipase A 2 (10). These effects of melittin result from its non- specific interaction with a wide variety of membranes (11- 16). A large number of studies have been undertaken to determine the conformational properties of melittin, with the aim of understanding the molecular mechanism of melittin’s interaction with membranes. Depending on the peptide concentration, pH, ionic strength, and the nature of the negative counterion, melittin is either monomeric in aqueous solution or associated as tetrameric aggregates (17-22). The aqueous monomer has no detectable secondary structure, as determined by circular dichroism (19, 23, 24) and 1 H NMR 1 (20). The X-ray structure of tetrameric melittin crystallized from aqueous solution (25) as well as the NMR structures of melittin in methanolic solution (26) and dodecylphospho- choline micelles (27-30) all indicate that the molecule consists of two R-helical segments (residues 2-10 and 13- 26). These segments are connected by a hinge (residues 11 and 12) to form a bent R-helical rod with the hydrophilic and hydrophobic sides facing opposite directions. The conformation of melittin when bound to perdeuterated dipalmitoylglycerophosphocholine vesicles, was determined ² This work was supported by grants from the U.S.A-Israel Binational Science Foundation (Grant 95-246 to J.A.) * To whom correspondence should be addressed. Phone: 972-8- 9343394. Fax: 972-8-9344136. ‡ Department of Structural Biology. § Department of Biological Chemistry. 1 Abbreviations: ATR-FTIR, attenuated total reflectance Fourier transform Infrared; CD, circular dichroism; COSY, 2D J-correlated spectroscopy; DMPG, 1,2 dimyristoyl-sn-glycerol-3-d54; DPC, dode- cylphosphocholine; HOHAHA, homonuclear Hartmann Hahn 2D experiment; NMR, nuclear magnetic resonance; NOE, nuclear Over- hauser enhancement; NOESY, 2D NOE spectroscopy; PC, egg phos- phatidylcholine; PG, egg phosphatidylglycerol; ROESY, rotating-frame Overhauser enhancement spectroscopy; RP-HPLC, reversed-phase high- performance liquid chromatography; SUV, small unilamellar vesicles; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy; TRNOE, transferred NOE; TSP, 3-(trimeth- ylsilyl) propionate, sodium salt; T 1F, spin-lattice, longitudinal relaxation time in the rotating frame; T2, spin-spin, transverse relaxation time; 2D, two-dimensional; 3D, three-dimensional. 15305 Biochemistry 1999, 38, 15305-15316 10.1021/bi991225t CCC: $18.00 © 1999 American Chemical Society Published on Web 10/26/1999