Strathprints logo
Strathprints Home | Open Access | Browse | Search | User area | Copyright | Help | Library Home | SUPrimo

Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation

Vita, C and Bontems, F and Bouet, F and Tauc, M and Poujeol, P and Vatanpour, H and Harvey, A L and Menez, A and Toma, F (1993) Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation. European Journal of Biochemistry, 217 (1). pp. 157-169. ISSN 0014-2956

Full text not available in this repository. (Request a copy from the Strathclyde author)


Charybdotoxin and two N-terminal truncated peptides, corresponding to the 2-37 and 7-37 sequences, were obtained by stepwise solid-phase synthesis using N alpha-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(II) 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 4.0-5.0%. Each protein was characterised chemically, structurally and functionally. 1H-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 three-dimensional 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(2+)-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 alpha/beta fold can be engineered with only 31 amino acid residues.

Item type: Article
ID code: 31666
Keywords: amino acid sequence, animals, brain, calcium, cell line, charybdotoxin, circular dichroism, disulfides, dogs, glutathione, kidney, magnetic resonance spectroscopy, molecular sequence data, peptide fragments, potassium channels, protein folding, rats, scorpion venoms, ultraviolet spectrophotometry, synaptosomes, Pharmacy and materia medica, Biochemistry
Subjects: Medicine > Pharmacy and materia medica
Department: Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences
Depositing user: Pure Administrator
Date Deposited: 13 Jul 2011 08:56
Last modified: 10 Dec 2015 20:08

Actions (login required)

View Item View Item