The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ([13Cδ1]-L-Trp) and glycine ([13Cα]Gly) were incorporated into melittin and three analogues by de novo peptide synthesis. 13C-Labeled tryptophan was incorporated into melittin at position 19 and into single-tryptophan analogues of melittin at positions 17, 11, and 9, respectively. Each of the synthetic peptides contained 13C-labeled glycine at position 12 only. The peptides were characterized functionally in a cytolytic assay, and spectroscopically by CD, fluorescence, and NMR. The behavior of 13C-labeled synthetic melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. All of the analogues were found to be efficient lytic agents and thus were functionally similar to the native peptide, yet no evidence was found for formation of a melittin-like tetramer by any of the analogues in aqueous media, although there was a propensity for apparently nonspecific peptide aggregation, especially for MLT-W9. Since the analogues did exhibit fractional helicities by CD comparable to or even greater than melittin itself in the presence of methanol, we infer that tetramer assembly requires not only the ability to form α-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 Cα was found to be a sensitive marker for helix formation in all of the peptides. For melittin itself, 13C NMR spectra revealed a downfield shift of ~ 1.8 ppm for the Gly-12 13Cα resonance of the tetramer relative to that observed for the free monomer in D2O. In mixed samples containing melittin monomer and tetramer, two discrete Gly-12 13Cα peaks were observed simultaneously, suggestive of slow exchange between the two species. We conclude that melittin's ability to form a soluble tetramer is not a prerequisite for cytolytic activity, nor is cytolytic potential precisely correlated with the ability to form an amphiphilic helix.
ASJC Scopus subject areas