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

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-dH containing d-amino acids [dV5, dV8, dL11 and dK16] 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-dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D2O demonstrated a large difference in dynamics between Mel-H and Mel-dH, whereby almost all backbone protons of Mel-dH showed a much faster rate of exchange as compared to Mel-H. Proton T1 relaxation had suggested a mobile backbone of Mel-dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-dH 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.