kalata-b1 has been researched along with Hemolysis* in 11 studies
11 other study(ies) available for kalata-b1 and Hemolysis
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Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies.
Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets. Topics: Amino Acid Sequence; Cell Survival; Cucurbitaceae; Cyclotides; Erythrocytes; HeLa Cells; Hemolysis; Humans; Magnetic Resonance Spectroscopy; Plant Proteins; Protein Structure, Tertiary | 2017 |
A Synthetic mirror image of kalata B1 reveals that cyclotide activity is independent of a protein receptor.
Featuring a circular, knotted structure and diverse bioactivities, cyclotides are a fascinating family of peptides that have inspired applications in drug design. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anti-cancer, anti-HIV, and hemolytic activities. In all of these activities, cell membranes appear to play an important role. However, the question of whether the activity of cyclotides depends on the recognition of chiral receptors or is primarily modulated by the lipid-bilayer environment has remained unknown. To determine the importance of lipid membranes on the activity of the prototypic cyclotide, kalata B1, we synthesized its all-D enantiomer and assessed its bioactivities. After the all-D enantiomer had been confirmed by (1)H NMR to be the structural mirror image of the native kalata B1, it was tested for anti-HIV activity, cytotoxicity, and hemolytic properties. The all-D peptide is active in these assays, albeit with less efficiency; this reveals that kalata B1 does not require chiral recognition to be active. The lower activity than the native peptide correlates with a lower affinity for phospholipid bilayers in model membranes. These results exclude a chiral receptor mechanism and support the idea that interaction with phospholipid membranes plays a role in the activity of kalata B1. In addition, studies with mixtures of L and D enantiomers of kalata B1 suggested that biological activity depends on peptide oligomerization at the membrane surface, which determines affinity for membranes by modulating the association-dissociation equilibrium. Topics: Anti-HIV Agents; Circular Dichroism; Cyclotides; Erythrocytes; Hemolysis; Humans; Kinetics; Lipid Bilayers; Stereoisomerism; Surface Plasmon Resonance | 2011 |
Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity.
Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids. Topics: Animals; Antinematodal Agents; Cyclotides; Cystine Knot Motifs; Erythrocytes; Gastrointestinal Tract; Haemonchiasis; Haemonchus; Hemolysis; Humans; Larva; Lysine; Mutagenesis, Site-Directed; Sheep; Trichostrongylosis; Trichostrongylus | 2010 |
Engineering stabilized vascular endothelial growth factor-A antagonists: synthesis, structural characterization, and bioactivity of grafted analogues of cyclotides.
Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclized backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Taking advantage of this stable framework we developed novel VEGF-A antagonists by grafting a peptide epitope involved in VEGF-A antagonism onto the stable cyclotide framework. Antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. A grafted analogue showed biological activity in an in vitro VEGF-A antagonism assay at low micromolar concentration and the in vitro stability of the target epitope was markedly increased using this approach. In general, the stabilization of bioactive peptide epitopes is a significant problem in medicinal chemistry and in the current study we have provided insight into one approach to stabilize these peptides in a biological environment. Topics: Chemistry, Pharmaceutical; Cyclotides; Cystine Knot Motifs; Drug Delivery Systems; Drug Design; Epitopes; Hemolysis; Humans; Hydrogen-Ion Concentration; Models, Chemical; Molecular Conformation; Neovascularization, Pathologic; Protein Structure, Tertiary; Vascular Endothelial Growth Factor A | 2008 |
Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis.
Cyclotides are macrocyclic plant peptides characterized by a knotted arrangement of three disulfide bonds. They display a range of interesting bioactivities, including anti-HIV and insecticidal activities. More than 100 different cyclotides have been isolated from two phylogenetically distant plant families, the Rubiaceae and Violaceae. In this study we have characterized the cyclotides from Viola yedoensis, an important Chinese herb from the Violaceae family that has been reported to contain potential anti-HIV agents. From V. yedoensis five new and three known cyclotides were identified and shown to have anti-HIV activity. The most active of these is cycloviolacin Y5, which is one of the most potent of all cyclotides tested so far using in vitro XTT-based anti-HIV assays. Cycloviolacin Y5 is the most hydrophobic of the cyclotides from V. yedoensis. We show that there is a positive correlation between the hydrophobicity and the anti-HIV activity of the new cyclotides and that this trend tracks with their ability to disrupt membranes, as judged from hemolytic assays on human erythrocytes. Topics: Amino Acid Sequence; Animals; Anti-HIV Agents; Australia; Cyclotides; Cystine Knot Motifs; Drugs, Chinese Herbal; Erythrocytes; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Structure; Plants, Medicinal; Protein Conformation; Rabbits; Sequence Homology, Amino Acid; Structural Homology, Protein; Viola | 2008 |
The cyclotide fingerprint in oldenlandia affinis: elucidation of chemically modified, linear and novel macrocyclic peptides.
The complete suite of cyclotides present in Oldenlandia affinis (Rubiaceae), the plant that was originally found to contain this unique family of circular proteins, has been characterised. This study expands the number of known cyclotides in this plant to 17, of which nine new sequences (kalata B9-B17) were characterised in this work. In addition, five derivatives that contain oxidation products of the conserved tryptophan were identified, and it was shown that the formation of these derivatives is catalysed by exposure to sunlight. Furthermore, we describe two "linear" cyclotide analogues. These acyclic peptides have three intact disulfide bonds, and their N and C termini coincide with the hypothesised cleavage sites from the precursor protein. This work increases our knowledge about the sequence variation that is accommodated by the cyclic cystine knot scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for cyclotides. These pathways have important implications for the persistence and environmental fate of the cyclotides if used as crop-protection agents. Topics: Acetylation; Alkylation; Amino Acid Sequence; Amino Acids; Chromatography, High Pressure Liquid; Cyclotides; Disulfides; Hemolysis; Lysine; Macrocyclic Compounds; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Oldenlandia; Oxidation-Reduction; Peptide Mapping; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Tryptophan | 2007 |
Cycloviolacin H4, a hydrophobic cyclotide from Viola hederaceae.
Cycloviolacin H4, a new macrocyclic miniprotein comprising 30 amino acid residues, was isolated from the underground parts of the Australian native violet Viola hederaceae. Its sequence, cyclo-(CAESCVWIPCTVTALLGCSCSNNVCYNGIP), was determined by nanospray tandem mass spectrometry and quantitative amino acid analysis. A knotted disulfide arrangement, which was designated as a cyclic cystine knot motif and characteristic to all known cyclotides, is proposed for stabilizing the molecular structure and folding. The cyclotide is classified in the bracelet subfamily of cyclotides due to the absence of a cis-Pro peptide bond in the circular peptide backbone. A model of its three-dimensional structure was derived based on the template of the homologous cyclotide vhr1 (Trabi et al. Plant Cell 2004, 16, 2204-2216). Cycloviolacin H4 exhibits the most potent hemolytic activity in cyclotides reported so far, and this activity correlates with the size of a surface-exposed hydrophobic patch. This work has thus provided insight into the factors that modulate the cytotoxic properties of cyclotides. Topics: Amino Acid Sequence; Amino Acids; Australia; Cyclotides; Erythrocytes; Hemolysis; Humans; Models, Molecular; Molecular Sequence Data; Protein Conformation; Sequence Alignment; Viola | 2006 |
Capped acyclic permutants of the circular protein kalata B1.
The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and C-termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity. Topics: Acetylation; Amino Acid Sequence; Animals; Cyclotides; Cysteine; Disulfides; Dose-Response Relationship, Drug; Erythrocytes; Hemolysis; Molecular Conformation; Molecular Sequence Data; Mutation; Nuclear Magnetic Resonance, Biomolecular; Plant Proteins; Protein Conformation; Protein Folding; Proteins; Rubiaceae; Sheep | 2004 |
Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity.
Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(24-28)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization. Topics: Amino Acid Sequence; Conserved Sequence; Cyclization; Cyclotides; Dose-Response Relationship, Drug; Hemolysis; Melitten; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides, Cyclic; Protein Structure, Secondary | 2003 |
An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides.
Four macrocyclic cystine-knot peptides of 29-31 residues, kalata, circulin A and B (CirA and CirB), and cyclopsychotride, have been isolated from coffee plants but have undetermined physiological functions. These macrocycles and 10 of their analogs prepared by chemical synthesis were tested against nine strains of microbes. Kalata and CirA were specific for the Gram-positive Staphylococcus aureus with a minimum inhibition concentration of approximately 0.2 microM. They were relatively ineffective against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. However, CirB and cyclopsychotride were active against both Gram-positive and Gram-negative bacteria. In particular, CirB showed potent activity against E. coli with a minimum inhibitory concentration of 0.41 microM. All four cyclic peptides were moderately active against two strains of fungi, Candida kefyr and Candida tropicalis, but were inactive against Candida albicans. These macrocycles are cytotoxic and lysed human red blood cell with a lethal dose 50% of 400 microM. Modifying the Arg residue in kalata with a keto aldehyde significantly reduced its activity against S. aureus whereas blocking the arg in CirA produced no significant effect. The two-disulfide variants and their scrambled disulfide isomers exhibited antimicrobial profiles and potency similar to their native peptides. However, in high-salt assays (100 mM NaCl), few of these macrocyclic peptides, natives or analogs, retained antimicrobial activity. These results show that the macrocyclic peptides possess specific and potent antimicrobial activity that is salt-dependent and that their initial interactions with the microbial surfaces may be electrostatic, an effect commonly found in defensin antimicrobial peptides. Furthermore, their end-to-end cyclic structure with a cystine-knot motif represents a molecular structure of antimicrobials and may provide a useful template for the design of novel peptide antibiotics. Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antifungal Agents; Candida; Cell Survival; Coffee; Computer Simulation; Cyclotides; Cystine; Disulfides; Erythrocytes; Escherichia coli; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Humans; Microbial Sensitivity Tests; Models, Molecular; Molecular Sequence Data; Peptides, Cyclic; Protein Conformation; Pseudomonas aeruginosa; Staphylococcus aureus | 1999 |
Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1.
Kalata B1 is a member of a new family of polypeptides, isolated from plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However, in the synthesis involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution. In addition to facilitating folding to a compact stable structure, cyclization has an important effect on biological activity as assessed by hemolytic activity. Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Circular Dichroism; Cyclization; Cyclotides; Cystine; Erythrocytes; Hemolysis; Humans; Mass Spectrometry; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Peptides, Cyclic; Plant Proteins; Plants, Medicinal; Protein Conformation; Protein Folding; Protein Precursors | 1999 |