Conformational Changes of Alamethicin Induced by Solvent and Temperature

Abstract

I3C nuclear magnetic resonance (NMR) and circular dichroism (CD) have been used for studies on the conformation of alamethicin. The 13C NMR spectrum is assigned with the aid of signals of synthetic partial sequences and selective proton decoupling. The solvent and temperature-dependence of the I3C NMR spectra, TI measurements and the use of lanthanide-shift reagents allow the differentiation between the amino acids belonging to a rigid a-helical portion of the alamethicin sequence and those belonging to a more flexible part. The 13C NMR results are in agreement with results obtained from extended solvent and temperature-dependent CD studies which indicate a highly stabilized nonpolar and intrachenar a-helical part. The concentration-dependence of the CD spectrum of alamethicin in a nematic phase revealed aggregation phenomena which might simulate those observed in natural and synthetic membranes. After dissolving alamethicin in aqueous alcohol there is a time-dependence of the ellipticity of the Cotton effects showing a sort of memory effect on the mode of dissolution. Four different conformations can be characterized by CD spectra depending on the solvent and concentration. A model illustrating the dynamic conformations and aggregation phenomena within a membrane is proposed.Alamethicin, a membrane active peptide (antibiotic U-22324) can be isolated from the culture broth of the fungus Trichoderma viride [ 11. Biosynthetic studies [2] suggest a non-ribosomal enzymic mechanism for the synthesis of this unusual sequence. The verylipophilic polypeptide has only one ionized group in the form of a glutamine carboxylic group. In addition to a y-glutamyl peptide bond there are seven residues of a-aminoisobutyric acid which contribute to the characteristic properties of alamethicin. The primary structure of a cycle which comprises 17 amino acids was determined in 1970 [3] and confirmed by mass spectrometry [4].The peptide has strong lysing properties on membranes of Ehrlich ascites carcinoma cells and on erythrocytes. These appear within very narrow ranges Abbreviations. CD, circular dichroism; NMR, nuclear magnetic resonance; Aib, a-amino-isobutyric acid; Yb(fod)3, 1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionato-ytterbium. .. show that alamethicin has a tendency to aggregate in neutral aequeous medium and that it might undergo hydrophobic interactions in phospholipids [12]. The tendency to form micelles at the lipid/water interface is also the basis of the models suggested so far to explain the molecular mechanism of the action of alamethicin on membranes [8,19]. The experiments exclude definitely a mobile carrier mechanism and point towards the formation of a hydrophilic ion-transporting "structure" in the membrane. So far ideas on the molecular structure of these membrane pores have been based on molecular model building. There have been no spectroscopic investigations on alamethicin, with the exception of a CD study [23,24] and a 60-MHz 'H NMR spectrum [13]. Therefore we have been carrying out conformatio...

Synthesis of a 19-residue peptide with alamethicin-like activity.

“…1). The former, proposed by Payne et al (19), has been confirmed by Ovchinnikov et al (20), while the latter, proposed by Martin and Williams (21,22), found the support of the Jung group (17 ALA-o, on the other hand, the same side group is engaged in an amide bond with phenylalaninol and the imino function of Pro2 is blocked by an Ac-Aib residue. Gln'9 again is identical in both sequences and provides the a-carboxyl group that makes ALA a monobasic acid.…”

“…First, in partial hydrolysates of ALA, a peptide comprising -Glu(y-Pro)-, the cycle-forming link, was not found (19). Second, the presence of blocking groups on the Glul' y-carboxyl (phenylalaninol) and the Pro2 imino group (Ac-Aib) was demonstrated (21,22,17), thus precluding a direct covalent link between these two residues. On these grounds, the synthesis of ALA-o rather than ALA-c was undertaken first.…”

“…Despite a number of physicochemical (13,14), conformational (15)(16)(17)(18), and chemical (17)(18)(19)(20)(21)(22) studies, the primary structure of ALA remains unknown. Currently, there are two proposed sequences: a cyclic (ALA-c) and an open chain (ALA-o) peptide structure (Fig.…”

600 MHz proton magnetic resonance studies of natural and synthetic alamethicin

“…In fact, the torsional angles, 4~ for Ala 6, Gln 7 and Val 9 are in the correct range for a helix (60°). From CD studies in methanol [13], it was estimated that 40% of the ALA backbone was in a helix. This degree of helicity is consistent with the observation that the 8 NHs in the segment from Aib3-Aib 1° exchange 'very slowly', and that residues 6, 7 and 9 have helical Q-values.…”

“…aH-NMR studies of partially purified Ala at 270 MHz [5] were instrumental in establishing its open-chain structure and the presence of the hitherto unsuspected phenylalaninol residue (see also [ 13]). The extent to which the spectra could be analyzed was limited by the low field and low resolution.…”