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

C Vita, F Bontems, F Bouet, M Tauc, P Poujeol, H Vatanpour, A L Harvey, A Ménez, F Toma

Research output: Contribution to journalArticle

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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 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.
LanguageEnglish
Pages157-169
Number of pages13
JournalEuropean Journal of Biochemistry
Volume217
Issue number1
DOIs
Publication statusPublished - 1 Oct 1993

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Charybdotoxin
Pyrrolidonecarboxylic Acid
Bioactivity
Biological Toxins
Proteins
High pressure liquid chromatography
Amino Acids
Solid-Phase Synthesis Techniques
Mercury
Nuclear magnetic resonance spectroscopy
Glutathione
Cysteine
Protons
Rats
Brain
Magnetic Resonance Spectroscopy
Tuning
High Pressure Liquid Chromatography
Nuclear magnetic resonance
Dogs

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

Cite this

Vita, C ; Bontems, F ; Bouet, F ; Tauc, M ; Poujeol, P ; Vatanpour, H ; Harvey, A L ; Ménez, A ; Toma, F. / Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation. In: European Journal of Biochemistry . 1993 ; Vol. 217, No. 1. pp. 157-169.
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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 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.",
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Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation. / Vita, C; Bontems, F; Bouet, F; Tauc, M; Poujeol, P; Vatanpour, H; Harvey, A L; Ménez, A; Toma, F.

In: European Journal of Biochemistry , Vol. 217, No. 1, 01.10.1993, p. 157-169.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Synthesis of charybdotoxin and of two N-terminal truncated analogues. Structural and functional characterisation

AU - Vita, C

AU - Bontems, F

AU - Bouet, F

AU - Tauc, M

AU - Poujeol, P

AU - Vatanpour, H

AU - Harvey, A L

AU - Ménez, A

AU - Toma, F

PY - 1993/10/1

Y1 - 1993/10/1

N2 - 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.

AB - 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.

KW - amino acid sequence

KW - animals

KW - brain

KW - calcium

KW - cell line

KW - charybdotoxin

KW - circular dichroism

KW - disulfides

KW - dogs

KW - glutathione

KW - kidney

KW - magnetic resonance spectroscopy

KW - molecular sequence data

KW - peptide fragments

KW - potassium channels

KW - protein folding

KW - rats

KW - scorpion venoms

KW - ultraviolet spectrophotometry

KW - synaptosomes

U2 - 10.1111/j.1432-1033.1993.tb18231.x

DO - 10.1111/j.1432-1033.1993.tb18231.x

M3 - Article

VL - 217

SP - 157

EP - 169

JO - European Journal of Biochemistry

T2 - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

IS - 1

ER -