The Interactions with Solvent, Heat Stability, and 13C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide

The peptide alamethicin was labelled with 13C and 15N by growing the fungus Trichoderma viride in a medium containing [U‐13C]glucose and K15NO3, Spin‐echo difference spectroscopy showed that13C was incorporated to a level of about 50% and15N to about 98%. Incorporation of 13C into the peptide provided residue‐specific probes of the interactions with solvent and heat stability of this ion‐channel‐forming peptide. All of the carbonyl carbons and the α‐carbons of the α‐aminoisobutyric acid [Ala(Me)J residues of alamethicin in methanol were assigned using two‐dimensional and three‐dimensional hetero‐nuclear correlation experiments. Measurements of 1Jc,N revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Glyl1 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. Chem. Soc. 117, 405–4101 that intramolecular hydrogen bonds in proteins and peptides are weaker than hydrogen bonds to solvent. The sensitivity of alamethicin carbonyl and proton chemical shifts to perturbation by dimethyl sulfoxide correlates well with the calculated solvent accessibilities of the carbonyls in the crystal structures and reveals residues in the middle of the peptide and at the C‐terminus which interact with solvent. Taken together with the 1Jc, measurements, the data support a model in which hydrogen bonding to solvent at the Gly1l/Leu12 amide could provide a site of hydration in the interior of the alamethicin channel structure. The temperature dependencies of the carbonyl chemical shifts support the suggestion that the peptide is flexible in the regions where solvent interacts with the backbone of the peptide. The linear temperature dependence of the carbonyl chemical shifts and molar ellipticity indicate that, due to steric constraints at the Ala(Me) residues, the peptide folding/folding transition is non‐cooperative and that the peptide is remarkably heat stable.