Characterization of selectively carbon-13-labeled synthetic melittin and melittin analogs in isotropic solvents by circular dichroism, fluorescence, and NMR spectroscopy

Abstract

The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ( [13C delta 1]-L-Trp) and glycine ( [13C alpha]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 alpha-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 C alpha 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 approximately 1.8 ppm for the Gly-12 13C alpha 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 13C alpha 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.

Determination of the secondary structure of selected melittin analogues with different haemolytic activities

“…These results correlate with the inability of these analogues to adopt an a-helical structure, and ultimately its tetrameric self-assembly, on increasing salt con' centration. Similar results, lower haemolytic activity and lower a-helical content than melittin, have been reported by Weaver et al (1989) for the double-substitution melittin analogue (subL9W + subWl9L). The c.d.…”

“…Fractional helicities (fH) were calculated as reported by Weaver et al (1989), and the secondary-structure content was estimated by a curve-fitting procedure (Yang et al, 1986 Quay and Condie (1983), 6280 = 5570 M-l cm-'. The absorption coefficients 6280 = 5570 M-l cm-' and 6280 = 11140 M-1 cm-1 were derived from these experiments, assuming an experimental error lower than 20%, and used for the peptides containing one or two tryptophan residues respectively.…”

13C?-NMR assignments of melittin in methanol and chemical shift correlations with secondary structure

“…0925-2738/$10.00 9 1993 ESCOM Science Publishers B.V. Weaver et al, 1989;Lakowicz et al, 1990). The sequence is H2N-GIGAVs-LKVLT~0-TGLPA15-LISWIE0-KRKRQQ-NH 2 (Habermann, 1972).…”

“…The crystal structure of the tetrameric aggregate has been solved to 2/k resolution (Terwilliger and Eisenberg, 1982 a,b) and has been correlated with the monomeric a-helical structure described by molecular dynamics (Pastore et al, 1989). Melittin has also proven to be a useful model for controlled studies of protein folding as well as for peptide-lipid interactions (Ikura et al, 1991;Weaver et al, 1992;Wilcox and Eisenberg, 1992).…”

Distributions of fluorescence decay times for synthetic melittin in water-methanol mixtures and complexed with calmodulin, troponin C, and phospholipids

“…Intensity decays were measured using magic-angle polarization conditions. Synthetic melittin was prepared using standard procedures for solid-phase peptide synthesis, as described previously [37], followed by purification by reverse-phase HPLC on a Cs column. CaM was from bovine testes and was a gift from Professor Robert Steiner, University of Maryland, Baltimore County.…”