2D-NMR and ATR-FTIR study of the structure of a cell-selective diastereomer of melittin and its orientation in phospholipids

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,I¹⁷,K²¹-melittin, D-amino acids at positions V^5,8,I¹⁷,K²¹) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V^5,8,I¹⁷,K²¹-melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]- V^5,8,I¹⁷,K²¹-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,I¹⁷,K²¹-melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were done with both melittin and [D]-V^5,8,I¹⁷,K²¹- 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 α-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,I¹⁷,K²¹- 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,I¹⁷,K²¹melittin. 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.