Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.
A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.
Huys
Isabelle
I
Laboratory of Toxicology, University of Leuven, Leuven, Belgium.
Dyason
Karin
K
Waelkens
Etienne
E
Verdonck
Fons
F
van Zyl
Johann
J
du Plessis
Johan
J
Müller
Gert J
GJ
van der Walt
Jurg
J
Clynen
Elke
E
Schoofs
Liliane
L
Tytgat
Jan
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D012605:Scorpions; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C454815:parabutoxin 3 protein, Parabuthus transvaalicus