Abstract
The cobrotoxin, isolated from Taiwan cobra (Naja naja atra) venom, is a small, basic, all β-sheet protein consisting of a single polypeptide chain of 62-amino acid residues, cross-linked by 4 disulfide bonds. Cobrotoxin binds specifically to the nicotinic acetylcholine receptor on the postsynaptic membrane and thus blocks neuromuscular transmissions. We report the direct expression of this protein from its synthetic gene in Escherichia coli. The clone of cobrotoxin was built in a ZZ construct, a synthetic IgG-binding domain of protein A fused at its N-terminus. A soluble fusion protein was obtained with a yield of 12 mg/L (corresponding to 4 mg/L for the free form of cobrotoxin). The toxin moiety was folded in IgG column in optimal oxidoreducing condition. The purified and refolded recombinant cobrotoxin sample is further characterized by SDS–PAGE, circular dichroism, and multi-dimensional NMR spectroscopy. Both the optical and NMR spectroscopy clearly suggest that the conformation features of the recombinant and authentic cobrotoxin are very similar. The interaction between cobrotoxin and two peptide fragments that correspond to the segment residues 122-138 and 182-202 of the nicotinic acetylcholine receptor was investigated by NMR technology. According the results of chemical shift perturbation, these two peptides both bind to cobrotoxin but with different binding sites in toxin molecule. The methods presented here described the first study of a high-level expression system with a simple purification and refolding procedure of cobrotoxin. It provides us a unique opportunity for NMR to investigate the interactions between cobrotoxin and the acetylcholine receptor/peptides. Superoxide dismutases (SODs) defend cells against oxidative damage and control superoxide concentrations. Defects in SOD contribute to neurodegenerative diseases apparently through the breakdown of radical defense mechanisms. MnSOD from different species form either dimer or tetramer. Vibrio alginolyticus MnSOD have high sequence homology with human mitochondrial MnSOD. But instead of being a tetramer, Vibrio alginolyticus MnSOD exists in a dimeric from. In present study, we use circular dichroism spectroscopy, fluorescence spectroscopy, NMR, size exclusion chromatography and sedimentation velocity measurements to investigate the unfolding of this dimeric molecular in GdnHCl. The results reveal that a stable partially structured intermediate accumulates in the GdnHCl-induced unfolding pathway of this protein. The intermediate accumulate maximally in 1.5 M GdnHCl and exhibits characteristics of a molten globule-like state. Results of the size-exclusion chromatography and sedimentation velocity experiments suggest dissociation of the protein into monomers only occur in the late stage of the unfolding process. According the results obtained from those techniques, we conclude the unfolding of apo MnSOD is a non-two-states process.