¹⁵N and ³¹P Solid-State NMR Investigations on the Orientation of Zervamicin II and Alamethicin in Phosphatidylcholine Membranes

Abstract

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.

Solid‐state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes

“…However, when the bilayer hydrophobic thickness is reduced to 1,2-dicapryl-sn-glycero-3-phosphocholine (di-C10:0-PC) transmembrane alignments of zervamicin II are observed. In contrast, alamethicin, which is 20 amino acids long, adopts transmembrane alignments under all conditions tested [28,29]. The lengths of zervamicin II and alamethicin di¡er by four amino acids only.…”

“…Similarly, the uncharged 16 amino acid peptide antibiotic zervamicin II is too short to span POPC phospholipid membranes in a stable transmembrane fashion. This peptide, therefore, exhibits mostly in-plane alignments [29]. However, when the bilayer hydrophobic thickness is reduced to 1,2-dicapryl-sn-glycero-3-phosphocholine (di-C10:0-PC) transmembrane alignments of zervamicin II are observed.…”

“…[21,26]). In contrast, many hydrophobic sequences, including the channel-forming M2 domain of in£uenza A [21,27], the peptide antibiotic alamethicin [28,29] or the L 6X3 -helix of gramicidin A [23], adopt transmembrane alignments. Investigations of the viral protein Vpu of HIV-1 involving a multitude of labelled sites indicate an approx.…”

“…This corresponds to a calculated di¡erence in length of 96 A î . At the same time the thickness of the lipid bilayers has to be decreased by more than 10 A î (from POPC to di-C10:0-PC) in order to accommodate zervamicin II in a stable transmembrane fashion [29]. Clearly other factors, such as conformational details in membrane environments or the length of the terminal anchoring sequences, contribute [43].…”

“…Not only is the knowledge of these interactions important for a quantitative evaluation of functional models of peptide activity [8], the study of model sequences [16] has also helped during the rational design of experimental conditions where the alignment of naturally occurring peptides shows the lowest mosaic spread [29]. Well-ordered samples are important for several structural techniques including oriented solid-state NMR spectroscopy [21,23].…”