Synthesis of charybdotoxin and of two N‐terminal truncated analogues

Abstract

Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7 -37 sequences, were obtained by stepwise solid-phase synthesis using W-t-butyloxycarbonyl and benzyltype side-chain protection. While this strategy was generally useful, the S-acetamidomethyl protecting group used for the six cysteines was not completely stable under HF treatment and its subsequent removal by mercury(I1) treatment was neither complete nor devoid of side reactions. The completely deprotected native and truncated sequences were folded efficiently in the presence of glutathione and were finally purified by high-pressure liquid chromatography with overall yields of 4.0-5.096. Each protein was characterised chemically. structurally and functionally. 'H-NMR spectroscopy was used and a complete assignment of all the protons of the three synthetic proteins was achieved. NMR data show that synthetic charybdotoxin is indistinguishable from the natural protein. The two truncated proteins contain the same elements of secondary structure and a similar overall threedimensional structure, in agreement with circular dichroic measurements. The shortest analogue, however, may have local structural perturbations and/or higher flexibility. Biological activity on dog epithelial Ca'+-activated K' channels and on rat brain synaptosomal voltage-dependent K' channels show that synthetic charybdotoxin was as potent as the natural toxin on both channels. For both channels, deletion of the first amino acid, 5-oxoproline (pyroglutamic acid) decreased only slightly the potency of the inhibitor, while deletion of the entire 1-6 segment reduced potency much more. We conclude that the N-terminal region of charybdotoxin plays a functional role in tuning the toxin's biological activity but is not essential for the folding and stability of the structure. The structure of the shortest analogue represents an interesting example of how a well organised and stable alp fold can be engineered with only 31 amino acid residues.Protein toxins are used by venomous animals to subdue prey. These molecules, which generally have a molecular mass ranging between 2-12 kDa, are active towards different molecular targets [1-41 and are characterised by the presence of several disulfide bridges. Scorpion venoms contain a variety of proteins known to inhibit Na' and K' channels. Charybdotoxin, present as a minor component (= 0.2%) in the venom of the Israeli Lpiurus quinquestriatus scorpion, was the first discovered high-affinity inhibitor of high-conductance Ca'+-activated K' channels present in skelctal muscles [5, 61. proteins 11, 3, 4, 20, 211 with subtly different specificities and which therefore represent valuable tools in order to study diffcrcnt K' channels.We have recently reported the three-dimensional solution structure of charybdotoxin solved by two-dimensional NMR [20, 211 and the refined and detailed description of the sidechain organisation [22]. This analysis revealed the presence of a well ordered structure containing three antiparallel strands fo...

A Potassium‐Channel Toxin From the Sea Anemone Bunodosoma Granulifera, An Inhibitor for Kv1 Channels — Revision of the Amino Acid Sequence, Disulfide‐Bridge Assignment, Chemical Synthesis, and Biological Activity

“…2A). The presence of polymeric species, after either high HF or low-high HF cleavage, was also observed in our laboratory in the synthesis of other cysteine-rich sequences [41]. Overall yields of purified product (1 1.5 %) were, however, quite satisfactory.…”

Chemical synthesis and structural characterization of the RGD‐protein decorsin: A potent inhibitor of platelet aggregation

“…These findings contrast with the highly efficient and clean reoxidation of hirudin HVl and its NH2-terminal fragment 1-43 (Chatrenet & Chang, 1993;Chang, 1994Chang, , 1995; see also Thannhauser et al, 1997) or fragment 1-47 (De Filippis et al, 1995) or 1-41 (unpublished) of hirudin HM2. Similar fast, quantitative, and correct refolding has been described for other small disulfide-crosslinked proteins, such as charybdotoxin (37-residue chain, three disulfides) (Vita et al, 1993Pierret et al, 1995).…”

“…obs. ) or charybdotoxin (Vita et al, 1993Pierret et al, 1995) do not contain any Pro and oxidatively re-fold very efficiently. Our proposal of a participation of Pro isomerization in the oxidative refolding process of reduced decorsin remains to be experimentally verified by demonstrating a catalytic effect of proline isomerase (Lang & Schmid, 1988;Fischer & Schmid, 1990;Fischer, 1994).…”

“…It is of interest to recall the characteristics of the near-W CD spectrum of charybdotoxin (37-residue chain, three disulfides). The near-UV CD spectrum of charybdotoxin shows a broad negative disulfide absorption in the 250-300 nm region, with a figure for [e] of about -14,000 deg.cm2.dmol" at 265 nm (Vita et al, 1993;Pierret et al, 1995), in agreement with the left-handed chirality of the three disulfides (X3 angle values ranging from -84" to -91") observed in the NMR structure of charybdotoxin (PDB code 2CRD; Bontems et al, 1992). Moreover, it is of interest to observe that the overall shape of the near-W CD spectrum and molar ellipticity value at 265 nm of synthetic refolded decorsin ([e], = 12,120 deg .…”

Changing the Structural Context of a Functional β-Hairpin

“…Oxidized and reduced glutathione, trypsin, chymotrypsin, and bovine serum albumin were from Sigma Toxin ␣, from Naja nigricollis, was purified as described previously (31). Charybdotoxin (synthetic) was obtained as published (32). Denatured chimera, used in binding assays, was obtained by carboxymethylation (33) of the six cysteines after disulfide reduction of the chimera and purified by HPLC.…”