Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction.
In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.
Chang
L S
LS
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan. lschang@mail.nsysu.edu.tw
Lin
S R
SR
Yang
C C
CC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry*; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral / Q000737:chemistry / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000596:Amino Acids; D003039:Cobra Neurotoxin Proteins; D005978:Glutathione; D005976:Glutaral