Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain.
Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding and internalization, the light chain (LC) acts as a unique Zn(2+)-endopeptidase against a target protein in the exocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate (ANS) revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low-pH-induced structural (and by implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LC to low pH (4.7) did not, however, evoke the loss of Zn(2+) bound to its active site. Implications of these observations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzed the nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescence measurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetime components estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observations suggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it is likely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structural alteration. This conclusion was further supported by the near-UV circular dichroism spectra under two pH conditions.
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Department of Chemistry and Biochemistry and Center for Marine Science and Technology, University of Massachusetts, Dartmouth 02747, USA.
Singh
B R
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D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D009498:Neurotoxins / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry / Q000379:methods; D013816:Thermodynamics
D046911:Macromolecular Substances; D009498:Neurotoxins; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A