title: Role of neurotoxin associated proteins in the low pH induced structural changes in the botulinum neurotoxin complex.
abstract: Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of -1.8 × 10(5)° cm(2)/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the Tm for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA, 02747, USA.
authors.forename: Gowri
authors.initials: G
authors.lastname: Chellappan
authors.forename: Raj
authors.initials: R
authors.lastname: Kumar
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary
chemical_list: D046912:Multiprotein Complexes; D009498:Neurotoxins; D001905:Botulinum Toxins