title: Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.
abstract: Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N → I1 [Formula: see text] I2 [Formula: see text] U, with two intermediate states I1 and I2. BoNT/A loses almost all its secondary structure in 3.75 M urea (I1), yet it displays a native-like secondary structure in 5 M urea (I2). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I2, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7 M urea. Our results stress the importance of structural flexibility in the toxin's mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity.
authors.affiliation: a Department of Chemistry and Biochemistry , Botulinum Research Center, University of Massachusetts , Dartmouth , MA , 02747 , USA .
authors.forename: Raj
authors.initials: R
authors.lastname: Kumar
authors.forename: Roshan V
authors.initials: RV
authors.lastname: Kukreja
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Artem
authors.initials: A
authors.lastname: Zhmurov
authors.forename: Olga
authors.initials: O
authors.lastname: Kononova
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
authors.forename: Syed A
authors.initials: SA
authors.lastname: Ahmed
authors.forename: Valeri
authors.initials: V
authors.lastname: Barsegov
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D019274:Botulinum Toxins, Type A / Q000737:chemistry*; D006358:Hot Temperature; D056004:Molecular Dynamics Simulation; D011042:Poisons / Q000737:chemistry*; D011489:Protein Denaturation; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013816:Thermodynamics; D014508:Urea
chemical_list: D011042:Poisons; D014508:Urea; D019274:Botulinum Toxins, Type A