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

Abstract

Melittin is a naturally occurring hexacosa peptide which forms an amphiphilic helix in methanol, a random coil in water, and a tetramer of helices at basic pH or in the presence of a high salt concentration. The monomeric structure in methanol has been well characterized by proton NMR (Pastore et al. (1989) Eur. Biophys. J., 16, 363-367). In the present paper, chemical shifts of the backbone alpha-carbons of melittin in methanol were determined by mapping previously published alpha-proton shifts (Bazzo et al. (1988) Eur. J. Biochem., 173, 139-146), to natural abundance (1H)13C cross peaks appearing in the 2D heteronuclear multiple-quantum NMR spectrum. Changes in chemical shifts consequent to stepwise increases in the percentage of water in a mixed methanol/water solvent system were observed in similar spectra. The alpha-carbon shifts varied more smoothly than the corresponding alpha-proton shifts and were found to correlate with the transition from the helix to the random coil conformer in parallel with changes in the circular dichroism spectrum. Chemical shifts of this peptide are interpreted with regard to the current database of assignments in proteins of known 3D structure (Wishart et al. (1991) J. Mol. Biol., 222, 311-333). The N-terminal region of the peptide shows increased flexibility at lower methanol concentrations, as evidenced by the merger of the alpha-proton resonances of G1 (at 40% and 15% methanol) and G3 (at 15% methanol). Conformational exchange rates for G1 and G3 were estimated by comparison of the experimental spectra with simulated spectra and found to be as large as 4000 s-1 for G1 in 40% and 15% methanol and 600 s-1 for G3 in 15% methanol. Overall, these 1H and 13C chemical shift data support the description of monomeric melittin in methanol currently evolving in the literature and suggest a structure composed of a linked pair of helices with different structural stabilities, each of which experiences dynamical fraying at its free terminus.

The structure, molecular dynamics, and energetics of centrin–melittin complex

“…Consequently, the C‐terminal end of MLT is the most stable portion of the peptide and is presumably stabilized by hydrogen bonding interactions with its aqueous environment. These results are consistent with those by Dr. Prendergast group using NMR spectroscopy 45–47…”

A study of ion-neutral collision cross-section values for low charge states of peptides, proteins, and peptide/protein complexes

“…[65-67] In addition, helical character was previously used to explain the results for gas-phase MS fragmentation reactions and MALDI hydrogen/deuterium (H/D) exchange,[19, 68] and formation of melittin oligomers with helical character has been also observed in basic or high ionic strength solutions using NMR studies. [69]…”

A uniform field ion mobility study of melittin and implications of low‐field mobility for resolving fine cross‐sectional detail in peptide and protein experiments

“…This observation is consistent with solution‐phase fluorescence and NMR measurements, which indicate the transition from random coil to an α‐helix occurs at 80% v/v methanol and greater. In particular, Miura found that melittin existed with partial helical characteristics below 80% methanol and is fully α‐helical above 80% methanol, which was observed across a fairly elevated temperature range 298–333 K. Solution‐phase circular dichroism and HDX also indicate a structural transition occurs in solution at around 70–80% methanol . Gas‐phase HDX experiments in an ion trap have suggested that [M + 2H] 2+ exists as a single, slow‐exchanging conformer consistent with a compact structure .…”