melitten and dodecylphosphocholine

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 alpha-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.

Topics: Amino Acid Sequence; Amino Acid Substitution; Cytotoxins; Lipid Bilayers; Melitten; Methanol; Micelles; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phosphatidylglycerols; Phospholipids; Phosphorylcholine; Protein Conformation; Protein Structure, Secondary; Spectroscopy, Fourier Transform Infrared; Stereoisomerism; Trifluoroethanol; Water

In our previous paper we reported the conformation of melittin bound to perdeuterated dodecylphosphocholine micelles as studied by 1H NMR experiment and distance geometry calculation. No hydrogen bonds were taken into consideration explicitly in the calculation. However, mostly alpha-helical conformations were obtained as results of the calculation even with no explicitly assumed hydrogen bonds. In the present paper we refined the distance geometry calculation by incorporating hydrogen bonds suggested by the previous calculation. As a result, we obtained the conformation of melittin, which was consistent with both NMR data and the additional hydrogen bonding data. The alpha-helical rod in the refined conformation also has a kink at Thr-11 and Gly-12, but its bent angle is now a bit narrowly distributed in 135 degrees +/- 15 degrees. In the present study another distortion at Trp-19 and IIe-20 becomes conspicuous. The average root-mean-square displacement of atoms is now much smaller and is 1.5 A for all backbone atoms. In the present paper side chain conformations are also analyzed.

Topics: Deuterium; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Melitten; Micelles; Molecular Structure; Phosphorylcholine; Protein Conformation

Previously, the size and stoichiometry of mixed micelles of perdeuterated dodecylphosphocholine and melittin were characterized and the 1H NMR spin systems of most amino acid residues of micelle-bound melittin identified. One- and two-dimensional 1H-1H Overhauser experiments have now been used to obtain qualitative information on intramolecular proton-proton distances. These data show that the N-terminal and the C-terminal segments of melittin form two spatially distinct, compact domains; using lipid spin labels these could be located near the micelle surface. For the C-terminal domain a detailed conformation was determined by using the distance contraints from the Overhauser studies as input for a distance geometry algorithm.

Topics: Animals; Bee Venoms; Bees; Electron Spin Resonance Spectroscopy; Magnetic Resonance Spectroscopy; Melitten; Micelles; Models, Molecular; Phosphorylcholine; Protein Conformation; Spin Labels

Assignments have been obtained for most of the H-NMR lines of melittin bound to fully deuterated dodecylphosphocholine micelles by combined use of two-dimensional spin echo correlated spectroscopy and one-dimensional NMR methods. Nuclear Overhauser enhancement measurements showed that the mobility of the entire polypeptide chain is reduced by binding of melittin to the detergent micelle and that the amino-terminal and carboxy-terminal halves of the primary structure constitute separate, compact domains within the conformation of micelle-bound melittin. p2H titration experiments showed that the presence of positive charges on the four amino groups of melittin had little influence on the conformation of the micelle-bound polypeptide. Titration of tetrameric melittin with detergent provided evidence that melittin assumes similar conformations as a self-aggregated tetramer and as a monomer bound to micelles.

Topics: Bee Venoms; Choline; Colloids; Magnetic Resonance Spectroscopy; Melitten; Micelles; Phosphorylcholine; Protein Conformation