Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part A. Sequence and conformation

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin.A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalaninol have l-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular ¹³C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially α-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous media due to a longer α-helical part and higher number of aliphatic residues. A part of the α-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high α-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin. Part A. Sequence and conformation.

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.     

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

Three cases of hemolytic Streptococcus infection in guinea-pigs (author's transl)

Three cases of naturally occurring hemolytic streptococcus infection were detected in guinea-pigs which had been delivered from different sources to the Division of Animal Research, Faculty of Medicine, University of Tokyo, Tokyo, during the period from November, 1976, till February, 1977. The clinical manifestations of the infection were differentiated into two types; ie, the acute type with sepsis and pneumonia as main pathologic changes, and the chronic type with abscess formation of submaxillary lymph node. Streptococcus zooepidemicus was isolated from the conjunctiva of about 56% of these guinea-pigs. Healthy guinea-pigs housed with spontaneously infected ones in the same cage suffered from the infection, showing manifestation and pathologic changes similar to the spontaneous cases. Some of them, however, remained apparently healthy for about 2 months harboring the organisms in the conjunctiva or nasal cavity. It is presumed that such carriers become a source of the infection in nature.     

Helical kink and channel behaviour: a comparative study with the peptaibols alamethicin,trichotoxin and antiamoebin

Kinks or bends introduced in peptides and proteins by helical distorter residues such as proline, other imino acids and glycine, especially when these are in close proximity in the sequence, are increasingly recognized as playing an essential role in the gating of channel-forming peptides as well as of physiological ion channels. Peptaibols are useful simple models for the much more complex biological ion channels, especially voltage-gated ones. In this short review, we compare the monomeric structures of three selected peptaibols (alamethicin, trichotoxin and antiamoebin) that widely differ with regards their near-central kink angles and dipolar moment orientations. These structural features are then shown to be correlated to the different patterns of channel activity, both at the macroscopic and single-channel levels of investigation.Presented at the British Biophysical Society–Société Française de Biophysique joint meeting Ion channels: from Biophysics to disorders, held in May 2003, Rennes, France     

Incorporation kinetics in a membrane, studied with the pore-forming peptide alamethicin.

The reaction of fluorescence-labeled alamethicin with unilamellar phospholipid vesicles (DOPC and DMPC) has been investigated in a stopped-flow apparatus. Clearly single exponential time functions have been observed at temperatures above the phase transition of the bilayer. This can be interpreted in terms of an essentially one-step incorporation process. The pseudo first-order forward rate is found to be quite fast, falling in a range somewhat below the diffusion controlled upper bound. The data are quantitatively very well described on the basis of a simple mechanism. This comprises diffusion of peptide into the bilayer accompanied by a more or less slower change of the secondary structure. Aggregation of the incorporated molecules at higher concentrations is indicated to be comparatively rapid.