Circulin D

Several macrocyclic peptides ( approximately 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional structure of one of these novel peptides, cycloviolacin O1, using (1)H NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants.

Improvements were made in the high-performance liquid chromatographic (HPLC) method to obtain baseline separation of chromatographic peaks of structurally similar polypeptide components in bacitracin. The improved method uses a 30-cm-long stainless-stell column packed with muBondapak C18. The theoretical plates of the column are approximately 140,000 per meter for the bacitracin A peak. The resolution function between bacitracins B1 and B2 and that between bacitracins A and B2 have been improved 418 and 225%, respectively. The components of bacitracin, bacitracins A, B, C, D, E, F, and G, were fractionated by the countercurrent distribution technique. These components, together with Compound X, a compound separated on a carboxymethylcellulose column, and bacitracin F, obtained by degrading bacitracin A sample at neutral pH, were used to identify peaks in the HPLC chromatogram. Effects of processing methods used to reduce microbial contamination levels in bacitracin powders were evaluated. Heat treatment caused a significant loss of antimicrobial activity (35% reduction), bacitracins A, B1, and B2 were reduced by 37, 22, and 21%, respectively. A significant increase (2.8 times) of bacitracin F, an oxidative degradation compound, was show. Irradiation by 60Co at 1.8 Mrad caused no loss of potency nor change in any of the bacitracin components. Ethylene oxide treatment, on the other hand, caused considerable (46%) reduction of potency. Substantial reduction of areas under the peak of bacitracins A, B1, and B2 (50, 24 and 37%, respectively) were noted. The chromatograms showed numerous unresolved peaks around bacitracins A, B1 and B2,; however, no significant increase in the bacitracin F peak, nor appearance of non-UV absorbing peaks were observed. Peptide antibiotics of the polymyxin group, circulin, colistin, and polymyxin, were also analyzed using the muBondapak C18 column with a linear-gradient elution. A UV monitor was used for polymyxin. A moving-wire flame ionization detector was used to monitor circulin and colistin. A sample of polymyxin, circulin, and colistin may be analyzed in less than 20 min of chromatographic time.