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Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

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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 × 105° cm2/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.

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Abbreviations

BoNT:

Botulinum Neurotoxin

CD:

Circular Dichroism

GI:

Gastrointestinal

LD:

Lethal dose

NAP:

Neurotoxin associated proteins

UV:

Ultra violet

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Acknowledgments

The authors would like to thank Steve Riding and Jenny Davis for preparing the different toxin proteins. This work was in part supported by a NIH Grant R03AI103868 and contract (HSHQDC-12-C-00071) from the Department of Homeland Security through Omni Array Biotechnology.

Conflict of interest

The authors declare that there are no conflicts of interest.

Ethical Standards

The experiments carried out in the study are in compliance with the current laws of the United States of America.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA, 02747, USA

    Gowri Chellappan & Shuowei Cai

  2. Botulinum Research Center, Institute of Advanced Sciences, 78-540 Faunce Corner Mall Road, North Dartmouth, MA, USA

    Raj Kumar & Bal Ram Singh

Authors
  1. Gowri Chellappan
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  2. Raj Kumar
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  3. Shuowei Cai
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  4. Bal Ram Singh
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Correspondence to Bal Ram Singh.

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Chellappan, G., Kumar, R., Cai, S. et al. Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex. Protein J 33, 557–564 (2014). https://doi.org/10.1007/s10930-014-9588-4

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  • DOI: https://doi.org/10.1007/s10930-014-9588-4

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