Spectroscopic Analysis of pH-Induced Changes in the Molecular Features of Type A Botulinum Neurotoxin Light Chain
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- 作者：Li Li and Bal Ram Singh
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：May 30, 2000
- 年：2000
- 卷：39
- 期：21
- 页码：6466 - 6474
- 全文大小：122K
- 年卷期：v.39,no.21(May 30, 2000)
- ISSN：1520-4995
文摘
Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitterrelease at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding andinternalization, the light chain (LC) acts as a unique Zn2+-endopeptidase against a target protein in theexocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed tothe endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding asindicated 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 (andby implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LCto low pH (4.7) did not, however, evoke the loss of Zn2+ bound to its active site. Implications of theseobservations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzedthe nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescencemeasurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetimecomponents estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observationssuggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it islikely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structuralalteration. This conclusion was further supported by the near-UV circular dichroism spectra under twopH conditions.