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 cycolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous stud y, a diastereomer ([D]-V-5,V-8,I-17,K-21-melittin, D-amino acids at positio ns V-5,V-8,I-17,K-21) of melittin was synthesized and its function was inve stigated [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; howev er, it retained antibacterial activity. Furthermore, [D]-V-5,V-8,I-17,K-21- melittin binds strongly and destabilizes only negatively charged phospholip id vesicles, in contrast to native melittin, which binds strongly also zwit terionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D] -V-5,V-8,I-17,K-21-melittin, and polarized attenuated total reflectance Fou rier transform infrared (ATR-FTIR) spectroscopy experiments were-done with both melittin and [D]-V-5,V-8,I-17,K-21-melittin. The structure of the dias tereomer was characterized by NMR in water, as well as in three different m embrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, meth anol, and dodecylphosphocholine/phosphatidylglycerol (DPC/DMPG) micelles. T he NMR data revealed an amphipathic alpha-helix only in the C-terminal regi on of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V-5,V-8,I-17, K-21-melittin are oriented parallel to the membrane surface. This study ind icates the role of secondary structure formation in selective cytolytic act ivity of [D]-V-5,V-8 ,I-17,K-21- melittin. While the N-terminal helical str ucture is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural eleme nt for binding and insertion into zwitterionic membranes and for hemolytic activity.