Chemical Synthesis and Folding Pathways of Large Cyclic Polypeptides:  Studies of the Cystine Knot Polypeptide Kalata B1

Abstract

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.

Acyclic Permutants of Naturally Occurring Cyclic Proteins

“…However, a significant proportion of correctly folded peptide was obtained from the linear precursor when oxidation reactions were performed in the presence of organic solvents. A hydrophobic solution environment appeared to be required to stabilize the surface-exposed hydrophobic residues that are formed upon folding into the native conformation (12). Furthermore, hemolytic activity was observed for cyclized kalata B1 but not for the oxidized linear derivative (12).…”

“…The circulins were found in a screen for anti-human immunodeficiency virus activity (8), cyclopsychotride A inhibits neurotensin binding to cell membranes (9), and kalata B1 shows uterotonic activity (6,10). Violapeptide I (11) was originally discovered based on its hemolytic activity, and it has recently been shown that kalata B1 (12,13), the circulins (12,13), and cyclopsychotride A (13) also have relatively weak hemolytic activity. A large number of additional peptides from this family have recently been discovered.…”

“…The three-dimensional structures of three members of this family (kalata B1 (10), circulin A (16), and cycloviolacin O1 (3)) have been determined by NMR spectroscopy and reveal the presence of a cyclic cystine knot motif, so named because of the N-to C-cyclized backbone and knotted three-dimensional structure (3,12). The disulfide bonds are in a cystine-knot arrangement with the Cys connectivities I-IV, II-V, and III-VI (10,17) in which two disulfide bonds and their connecting backbone segments produce an eight-residue cycle through which the third disulfide bond "threads" (10).…”

“…We have previously reported the synthesis and folding of kalata B1 using two separate strategies (12), one in which the disulfide bonds were formed before cyclization, and the other in which cyclization preceded oxidation of the cysteine residues. These two strategies allowed a comparison of the folding of kalata B1 in the linear and cyclic forms.…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…[4] It is believed that the compact CCK motif of cyclotides is responsible for their exceptional resistance against chemical, thermal, and proteolytic degradation. [5] Cyclotides display a diverse range of biological activities, including uterotonic, [6] haemolytic, [7][8][9] neurotensin antagonistic, [10] HIV inhibitory, [11] antimicrobial, [12] cytotoxic, [13] antifouling, [14] and trypsin inhibitory activities. [15] In addition, kalata B1 and B2 have been shown to inhibit the growth and development of Helicoverpa punctigera and H. armigera larvae, suggesting that cyclotides function as insecticidal agents as part of plant defence systems.…”