phalloidine and viroisin

NMR studies have revealed that the conformation of the monocyclic viroisin is dissimilar to that of the corresponding monocyclic derivative of phalloidin, dethiophalloidin, but has much similarity with the conformation of the bicyclic phalloidin. Obviously, one of three structural features found exclusively in the virotoxins is able to compensate for the conformational strain that in the bicyclic phallotoxins maintains the toxic conformation. Synthetic work on virotoxin analogues has shown that both the additional hydroxy group in allo-hydroxyproline and the methylsulfonyl moiety in the 2'-position of tryptophan are unlikely to represent the structural element in question, leaving the D-serine moiety as the supposed key element. In this study we asked whether it is the hydroxy group of this amino acid or its D-configuration that is responsible for the effect. We synthesized four viroisin analogues and submitted them to conformational analysis by NMR as well as to an actin binding assay. While the rotating-frame nuclear Overhauser effect (ROESY) spectra of the analogues with L-configured amino acids showed several sets of signals, indicating the existence of conformers interconverting more slowly than the NMR time scale, the spectra of the analogues with D-configured amino acids showed only one set of signals. Remarkably, the two viroisin analogues with D-serine and D-alanine also had distinctly higher affinities for filamentous actin than their L-configured counterparts, suggesting that the high biological activity may be correlated with the absence of multiple and slowly interconverting conformers. Anyhow, D-configuration of serine is the structural element that maintains the phalloidin-like structure, while the hydroxy group does not contribute to conformational stability but is likely to be in contact with the actin surface.

Topics: Actins; Alanine; Amino Acid Substitution; Animals; Binding Sites; Biological Assay; Nuclear Magnetic Resonance, Biomolecular; Peptides, Cyclic; Phalloidine; Protein Conformation; Rabbits; Serine; Structure-Activity Relationship

We investigated the conformations of toxic phalloidin and viroisin in aqueous solution using 500-MHz 1H-NMR spectroscopy in conjunction with molecular modeling. The conformations of two non-toxic phalloidin derivatives, secophalloidin and dethiophalloidin, were also correspondingly studied for comparison purposes. Results indicate that the non-toxic peptides have a multiple conformation, whereas the toxic peptides are comprised of a rigid molecule. It was found that the conformation of phalloidin partially resembles that of viroisin in the region of Cys3-Pro4-Ala5-Trp6, being different from that of the non-toxic peptides; thereby suggesting this region plays an important role leading to their toxicity.

Topics: Amino Acid Sequence; Carbon; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Peptides, Cyclic; Phalloidine; Protein Conformation; Protons; Solutions; Structure-Activity Relationship; Water

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.

Topics: Actins; Amoeba; Animals; Cell Membrane; Peptides, Cyclic; Phalloidine; Polymers

We have developed a new technique for the permeabilization of the membrane of Acanthamoeba castellanii. This technique involves the use of digitonin which alters neither the morphology nor the motility of the cell, but favours the penetration of phalloidin and viroisin. Treatment of permeabilized cells with phalloidin or viroisin induces, in the cortex of the cell, an intensive proliferation of filaments which have been identified as actin. This cortical filamentous layer detaches from the membrane and slowly contracts, acting as a fine mesh sieve which concentrates the organelles in the middle of the cell, causing therefore the formation of a central granuloplasm and a cortical hyaloplasm. During this process, cell motility is irreversibly lost. The results indicate that extensive proliferation and reorganization of actin filaments cannot support cell motility and they are discussed in terms of a general understanding of amoeboid movement.

Topics: Actin Cytoskeleton; Actins; Amoeba; Animals; Cell Membrane Permeability; Digitonin; Microscopy, Electron; Oligopeptides; Peptides, Cyclic; Phalloidine

Virotoxins are a group of monocyclic peptides recently identified in the deadly mushroom Amanita virosa by Faulstich and coll. We found that two of these peptides, which have a methyl sulfonyl group, namely viroidin and viroisin are very effective to protect F-actin against oxidative degradation by osmium tetroxide in vitro. Their desoxo analogs, which have a methyl sulfoxyde group instead of methyl sulfonyl are less active, therefore there exists a relationship between the chemistry of the sulfur group and the activity of the peptides.

Topics: Actins; Agaricales; Amanita; Animals; In Vitro Techniques; Osmium; Osmium Tetroxide; Peptides, Cyclic; Phalloidine; Rabbits