Sequences of alamethicins F30 and F50 reconsidered and reconciled

Abstract

From the culture broth of the mould Trichoderma viride, strain NRRL 3199, a microheterogeneous mixture of the membrane active 20-residue peptaibol alamethicin (ALM) could be isolated. ALMs were isolated by XAD-2 column chromatography and separated by silica gel chromatography and trichloromethane/MeOH gradient elution into an acidic and neutral group of peptides, named ALM F30 and ALM F50, respectively, according to their 100 Rf on TLC. Peptides ALM F50 were separated by semi-preparative and analytical HPLC and subjected to ESI-MS. Ten sequences of ALM F30 and their relative quantities could be determined. The major peptides ALM F30/3 (46%) and ALM F30/7 (40%), distinguished by Aib/Ala exchange in position 6, correspond to sequences described as ALM I and II occurring in the original alamethicin from Upjohn Company. Analogously, 13 sequences of the neutral peptide mixture named ALM F50 could be determined. The major peptide ALM F50/5 (75%) and the minor peptide ALM F50/7 (10%) are distinguished from ALM F30/3 and ALM F30/7 by having Gln17 in place of Glu17, the latter occurring in the F30 group. Notably. currently commercially available alamethicins (Fluka, Sigma) represent microheterogeneous mixtures of the neutral ALM F50 peptides with trace amounts of acidic ALM F30 peptides.

Crystal Structure of a Spin‐Labeled, Channel‐Forming Alamethicin Analogue

“…[1] Alamethicins, the longest peptaibols, are a group of closely sequence-related peptides composed of 19 amino acid residues. [2] They are able to form voltagedependent pores in biological membranes and are the most extensively investigated among simple model compounds of large pore-forming proteins.…”

Peptaibiomics: Towards a Myriad of Bioactive Peptides Containing Cα‐Dialkylamino Acids?

“…Sequences of Alamethicins Reconsidered and New Peptaibiotics from Paecilomyces. In 2003, the sequences of ALM F30 and ALM F50 were thoroughly reconsidered and reconciled [8]: HPLC separation of ALM F30 using an acidic gradient revealed ten individual sequences of ALM F30/1 -10. Basically, the same gradient was used to separate ALM F50 but omitting the addition of TFA to the eluents.…”

“…In 1967, the first report on the Aibcontaining antibiotic U-22324 from Trichoderma viride NRRL 3199 was published [34]. This polypeptide antibiotic, later renamed alamethicin (ALM), is generally regarded as the most thoroughly investigated peptaibiotic, thus often serving as the classical paradigm of a peptaibol [8] [10] [12] [35] [36]. The antiprotozoal activity of antiamoebin (AAM) was published later the same year [37].…”

“…Recently, the same material used by Kirschbaum et al [8] was further analyzed by nonaqueous capillary electrophoresis/mass spectrometry (NACE/MS): eleven amino acid sequences were identified, and characterized by the exchange of Ala to Aib in position 6, Gln to Glu in position 7 or 19. To continue, two novel ALM were detected, which were characterized by the loss of the C-terminal Pheol residue, thus terminating in Gln.…”

Peptaibiomics: Screening for Polypeptide Antibiotics (Peptaibiotics) from Plant-ProtectiveTrichoderma Species

“…The gradient used for HPLC and Ion-Trap-ESI-LC-MS measurements has been described in [3]; further details concerning the anal. equipment were given in [66].…”