tachyplesin-peptide--tachypleus-tridentatus and dodecylphosphocholine

Tachyplesin I is a 17-residue peptide isolated from the horseshoe crab, Tachypleus tridentatus. It has high antimicrobial activity and adopts a beta-hairpin conformation in solution stabilized by two cross-strand disulfide bonds. We report an NMR structural investigation of wild-type tachyplesin I and three linear derivatives (denoted TPY4, TPF4, and TPA4 in which the bridging cysteine residues are uniformly replaced with tyrosine, phenylalanine, and alanine, respectively). The three-dimensional aqueous solution structures of the wild type and the active variant TPY4 reveal very similar beta-hairpin conformations. In contrast, the inactive variant TPA4 is unstructured in solution. The arrangement of the tyrosine side chains in the TPY4 structure suggests that the beta-hairpin is stabilized by aromatic ring stacking interactions. This is supported by experiments in which the beta-hairpin structure of TPF4 is disrupted by the addition of phenol, but not by the addition of an equimolar amount of cyclohexanol. We have also determined the structures of wild-type tachyplesin I and TPY4 in the presence of dodecylphosphocholine micelles. Both peptides undergo significant conformational rearrangement upon micelle association. Analysis of the micelle-associated peptide structures shows an increased level of exposure of specific hydrophobic side chains and an increased hydrophobic integy moment. Comparison of the structures in micelle and aqueous solution for both wild-type tachyplesin I and TPY4 reveals two requirements for high antimicrobial activity: a beta-hairpin fold in solution and the ability to rearrange critical side chain residues upon membrane association.

Topics: Amino Acid Sequence; Amino Acid Substitution; Amino Acids, Aromatic; Animals; Antimicrobial Cationic Peptides; Crystallography, X-Ray; Cyclohexanols; DNA-Binding Proteins; Horseshoe Crabs; Micelles; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides, Cyclic; Phenol; Phosphorylcholine; Protein Conformation; Protein Isoforms; Protons; Solutions; Thermodynamics; Water

The mode of action of tachyplesin I, an antimicrobial cationic heptadecapeptide amide isolated from the hemocyte debris of a horseshoe crab, Tachypleus tridentatus, toward lipid matrices was studied with synthetic tachyplesin I, its analogs with Phe in place of Trp or Tyr, a linear analog with no disulfide bonds, and two linear short fragments. Circular dichroism spectra showed that tachyplesin I took an antiparallel beta-structure in buffer solution and a certain less ordered structure in acidic liposomes composed of egg phosphatidylcholine and egg phosphatidylglycerol (3:1). Spectrophotometric titration of the peptides with laurylphosphorylcholine revealed that both Trp and Tyr residues orient toward the inside of lipid matrices, suggesting that they are on the same side of the peptide backbone. The carboxyfluorescein leakage experiment and fluorescence data indicated that tachyplesin I interacted strongly with neutral and acidic lipid bilayers and an aromaticity-rich hydrophobic part of the peptide was embedded in lipid membranes. All the peptides except for the short fragments were almost equally active in lipopolysaccharide binding. The energy-transfer experiment showed that a conformational change occurred such that the Tyr and Trp residues are positioned more closely to each other in acidic liposomes than in buffer solution. The present study strongly suggested that amphipathic lipid bilayers induced a conformational change of tachyplesin I from an energetically stable beta-structure to a less ordered, probably more amphipathic structure.

Topics: Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Circular Dichroism; DNA-Binding Proteins; Fluoresceins; Lipids; Lipopolysaccharides; Liposomes; Molecular Sequence Data; Peptides; Peptides, Cyclic; Phosphorylcholine; Protein Conformation; Tryptophan; Tyrosine