Fractionation Protocol for the Isolation of Polypeptides from Plant Biomass

299

285

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Mö bius cyclotides) and knots (a knotted arrangement of the disulfide bonds).

Section: A Backbone Twist Caused By a Cis-peptide Bond Defines The Mösupporting

confidence: 90%

Section: Structural Roles Of Individual Amino Acids In the Cyclotidementioning

confidence: 99%

Twists, Knots, and Rings in Proteins

Rosengren

Daly

Plan

et al. 2003

299

285

“…Cyclic CS␤ antimicrobial peptides known as cyclotides have also been discovered in plants and animals (12)(13)(14)(15)(16). These end-to-end constrained peptides include the Rhesus theta defensin (RTD-1) that has been identified from Rhesus monkey leukocytes (17).…”

mentioning

confidence: 99%

Correlations of Cationic Charges with Salt Sensitivity and Microbial Specificity of Cystine-stabilized β-Strand Antimicrobial Peptides

Tam

Yang

2002

The electrostatic interaction of the charge cluster of an amphipathic peptide antibiotic with microbial membranes is a salt-sensitive step that often determines organism specificity. We have examined the correlation between charge clusters and salt insensitivity and microbial specificity in linear, cyclic, and retro-isomeric cystine-stabilized ␤-strand (CS␤) tachyplesin (TP) in a panel of 10 test organisms. Cyclic tachyplesins consisting of 14 and 18 amino acids are constrained by an end-to-end peptide backbone and two or three disulfide bonds to cross-brace the anti-parallel ␤-strand that approximates a "␤-tile" structure. Circular dichroism measurements of ␤-tile TPs showed that they displayed ordered structures. Control peptides containing the same number of basic amino acids as TP but lacking disulfide constraints were highly salt sensitive. Cyclic TP analogues with six cationic charges were more broadly active and salt-insensitive than those with fewer cationic charges. Reducing their proximity or number of cationic charges, particularly those with three or fewer basic amino acids, led to a significant decrease in potency and salt insensitivity, but an increased selectivity to certain Gram-positive bacteria. An end-group effect of the dibasic N-terminal Lys of TP in the open-chain TP and its retroisomer was observed in certain Gram-negative bacteria under high-salt conditions, an effect that was not found in the cyclic analogs. These results suggest that a stable folded structure together with three or more basic amino acids closely packed in a charged region in CS␤ peptides is important for salt insensitivity and organism specificity.

“…A large number of additional peptides from this family have recently been discovered. The varv peptides A-H have been isolated from Viola arvensis (14,15), and we have reported the discovery of several peptides from Viola odorata, Viola hederaceae, and Oldenlandia affinis (3). There is approximately 40% sequence conservation throughout the known sequences.…”

mentioning

confidence: 99%

Acyclic Permutants of Naturally Occurring Cyclic Proteins

Daly

Craik

2000

104

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple-stranded ␤-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the ␤-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.