title: Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: implications in cell selective lysis by D-amino acid containing antimicrobial peptides.
abstract: Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-(d)H containing d-amino acids [(d)V5, (d)V8, (d)L11 and (d)K16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-(d)H both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-(d)H interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-(d)H showed multiple beta-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D(2)O demonstrated a large difference in dynamics between Mel-H and Mel-(d)H, whereby almost all backbone protons of Mel-(d)H showed a much faster rate of exchange as compared to Mel-H. Proton T(1) relaxation had suggested a mobile backbone of Mel-(d)H peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-(d)H is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.
authors.affiliation: Biomolecular NMR and Drug Discovery Laboratory, School of Biological Sciences, Division of Structural and Computational Biology, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Rathi
authors.initials: R
authors.lastname: Saravanan
authors.forename: Anirban
authors.initials: A
authors.lastname: Bhunia
authors.forename: Surajit
authors.initials: S
authors.lastname: Bhattacharjya
mesh_terms: D000818:Animals; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008555:Melitten / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D008823:Micelles; D008555:Melitten