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

Abstract

The peptide alamethicin was labelled with I3C and "N by growing the fungus Trichoderma viride in a medium containing [U-"C]glucose and K"N0,. Spin-echo difference spectroscopy showed that I3C was incorporated to a level of about SO% and "N to about 98%. Incorporation of "C into the peptide provided residue-specific probes of the interactions with solvent and heat stability of this ion-channelforming peptide. All of the carbonyl carbons and the a-carbons of the a-aminoisobutyric acid [Ala(Me)J residues of alamethicin in methanol were assigned using two-dimensional and three-dimensional heteronuclear correlation experiments. Measurements of '.Ic,, revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Glyl 1 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. 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 'JC,, measurements, the data support a model in which hydrogen bonding to solvent at the GlylULeul2 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 foldinghnfolding transition is non-cooperative and that the peptide is remarkably heat stable. Keywords:alamethicin ; "C labelling; dimethyl sulfoxide ; ion channel ; NMR.Alamethicin is a 20-amino-acid antibiotic peptide secreted by the fungus Trichoderma viride. It forms a voltage-gated conductance in lipid bilayers (Eisenberg et al., 1973;Latore et al., 1981) and is one of the most extensively studied models for vertebrate ion channels [for reviews see Sansom (1993) and Cafiso (1994)l. The peptide has eight residues of a-aminoisobutyric acid [Ala(Me)] which have a-methyl in place of the usual a-hydrogen found in the L-amino acids. The mechanism by which alamethicin inserts into membranes, forms ion channels, and is regulated by an applied voltage is unclear. You et al.(1 996) recently showed that covalent dimers of alamethicin form stabilized ion channels with a single predominant conductance state. They suggest that the channels consist of a bundle of six parallel monomers. Evidence for antiparallel interactions between monomers dissolved in methanol was obtained by North et al. (1994). Their measurements of the paramagnetic enhancements of nuclear relaxation also indicated that the peptide backbone c...

Antiamoebin I in Methanol Solution: Rapid Exchange between Right‐Handed and Left‐Handed 310‐Helical Conformations

“…Moreover, the C¼O group of Gly 6 , and the amide H-atoms of Aib 8 and Aib 9 are exposed to solvent and form the site of a strong hydration in the middle of the molecule. The 1 J NC pattern for Aam-I is more similar to that observed in alamethicin [45] than to that in Zrv-IIB, where no significant backbone hydration was observed [39].…”

Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H?15N NMR spectroscopy

“…Chemical shift, ROE, and circular dichroism measurements on alamethicin suggest that the molecule is less flexible at lower temperatures (Esposito et al, 1987;Yee et al, 1996). However, the measured order parameters do not seem to be sensitive to this difference (Fig.…”

“…The dynamics of seven of the non-Aib c~-carbons in alamethicin and some of the side chains were investigated by natural abundance ~3C NMR spectroscopy by Kelsh et al (1992). Alamethicin is well-suited to relaxation-rate analysis, as it can be labelled with 15N (Yee and O'Neil, 1992) and 13C (Yee et al, 1996). To investigate the dynamics of alamethicin at both the L-amino acids and the Aib residues, we have measured residue-specific ~SN relaxation data for uniformly labelled alamethicin dissolved in methanol and in aqueous sodium dodecylsulfate (SDS) solution.…”

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

“…For example, the b-helical structures of gramicidin A fail to melt in octanol at temperatures up to 65 8C; [39] likewise, Seebach reported that the 3 14 -helical structures of a b 3 hexapeptide and a b 3 heptapeptide remained largely intact up to 60 8C in methanol, [42] and others have shown that 3 10 -helical peptides rich in the a,a-dialkylated residue a-aminoisobutyric acid (Aib) exhibit exceptional thermal stability. [40,41] The high thermal stability observed in these systems has been attributed to a dominance of steric effects over hydrogen bonding, and Wu et al have advanced a related explanation for the high thermal stability of b 3 -peptide 3 14 helices in particular. On the basis of theoretical calculations, these authors reported that, in contrast to a-helical peptides, the helical and extended forms of model b 3 peptides are similar in entropy, and, thus, no large entropic driving force exists for unfolding at elevated temperatures.…”