Changing the structural context of a functional beta-hairpin. Synthesis and characterization of a chimera containing the curaremimetic loop of a snake toxin in the scorpion alpha/beta scaffold

J Biol Chem. 1996 May 17;271(20):11979-87. doi: 10.1074/jbc.271.20.11979.

Abstract

An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure acting as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar beta-hairpin of the scorpion toxin charybdotoxin, stabilized by a short helix. The resulting chimeric molecule, only 31 amino acids long, was produced by solid phase synthesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected alpha/beta fold characteristic of scorpion toxins and presented a remarkable structural stability. The chimera competitively displaces the snake curaremimetic toxin alpha from the acetylcholine receptor at 10(-5) M concentrations. Antibodies, elicited in rabbits against the chimera, recognize the parent snake toxin and prevent its binding to the acetylcholine receptor, thus neutralizing its toxic function. All these data demonstrate that the strategy of active site transfer to the charybdotoxin scaffold has general applications in the engineering of novel ligands for membrane receptors and in vaccine design.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Charybdotoxin / chemistry*
  • Chromatography, High Pressure Liquid
  • Circular Dichroism
  • Cobra Neurotoxin Proteins / chemistry*
  • Molecular Sequence Data
  • Potassium Channel Blockers
  • Protein Folding
  • Protein Structure, Secondary
  • Rabbits
  • Receptors, Cholinergic / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / immunology

Substances

  • Cobra Neurotoxin Proteins
  • Potassium Channel Blockers
  • Receptors, Cholinergic
  • Recombinant Fusion Proteins
  • Charybdotoxin
  • alpha-cobratoxin