title: Conformational changes associated with the nicking and activation of botulinum neurotoxin type E.
abstract: Secondary and tertiary structural parameters of type E botulinum neurotoxin in the unactivated single-chain and activated two-chain (i.e., after proteolytic cleavage) forms were analyzed using circular dichroism, derivative absorption and fluorescence spectroscopy. The estimated secondary structures (22 and 20% alpha-helix, 44 and 44% beta-pleated sheets, and 34 and 36% random coils for the single- and two-chain neurotoxins, respectively) indicated that virtually no change occurred upon nicking of the single-chain neurotoxin. About 57% of the 70 Tyr residues were exposed in the single-chain form, which increased to 62% in the two-chain form. Fluorescence quenching experiments with neutral, anionic and cationic quenchers indicated that about 40% of the maximum accessible fluorescent Trp residues were exposed on the surface of the single-chain neurotoxin as compared to only 20% in the case of the two-chain neurotoxin. Acrylamide was the most effective quencher with a fraction accessibility of 0.56 and 0.48 of maximum accessible Trp fluorescence residues in the single and two-chain forms of the neurotoxin, respectively. Native polyacrylamide gel electrophoresis of the two forms of the neurotoxin revealed greater mobility for the two chain form. This indicates that the surface charges in the single-chain neurotoxin were altered upon nicking. These observations suggest that nicking of the single-chain type E neurotoxin results in refolding and redistribution of the surface charges of the neurotoxin.
authors.affiliation: Food Research Institute, University of Wisconsin, Madison 53706.
authors.forename: B R
authors.initials: BR
authors.lastname: Singh
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001905:Botulinum Toxins