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Structure and function relationship of botulinum neurotoxin and a neurotoxin associated protein

Fen-Ni Fu, University of Massachusetts Amherst


In this study, a structural role of the zinc in addition to its catalytic role in the type A botulinum neurotoxin has been examined. Both secondary and tertiary structure are altered upon the removal of the bound zinc. While the binding Zn$\sp{2+}$ seems to be reversible, the structure and in biological activity of the type A botulinum neurotoxin were irreversible by a altered upon removal of Zn$\sp{2+}$. Botulinum neurotoxin is internalized into neuronal cells through receptor-mediated endocytosis. Lowering of endosomal pH has been proposed to trigger the translocation of whole or a part of the neurotoxin, possibly through a membrane channel formation by the neurotoxin's heavy chain subunit. Low pH-induced voltage gated channels were observed only with the heavy chain and not with the intact neurotoxin. In liposomes membrane channel activity, as monitored by calcein release, was observed with the intact neurotoxin as well as its two subunit chains. The heavy chain exhibited the highest Calcein release activity. The pH effect on the membrane channel formation by botulinum neurotoxin was therefore, investigated to understand the molecular basis of membrane channel formation of heavy chain. The low pH induced significant conformational change in the botulinum neurotoxin heavy chain at the tertiary structure level, which could be responsible for triggering its insertion into membrane binding. The secondary structure of the light chain was dramatically changed, which could explain its interaction with membrane lipid as well as its likely passage through the membrane channel formed by the heavy chain. Low pH-induced changes in polypeptide folding were also analyzed by monitoring thermal unfolding of the heavy chain in different pH conditions. Thermal unfolding curves indicated the presence of oligomeric structure at low pH. Binding of the heavy chain with liposomes at either pH 7.4 or 4.0 introduced dramatic changes in the unfolding pattern, suggesting major structural alternation in the protein upon binding with membrane. The heavy-chain-induced liposome aggregation as well as fusion, which could be relevant to the possible oligomeric structure of the neurotoxin and the presence of the neurotoxin translocation. With the use of specific antibodies, we examined the role of specific peptide segments of heavy chain in the membrane channel activity. During the isolation of the neurotoxin, 33 kDa protein is serendipitously purified from the botulinum neurotoxin complex. This protein is an integral part of a group of neurotoxin associated proteins which are known to protect the neurotoxin from adverse environmental conditions, including acidity and protease of the gastric juice. The purified protein exhibited hemagglutination activity which was strongly inhibited by sugars. The protein was also completely resistant to the trypsin digestion. Structural investigation indicated the hemagglutinin-33 protein is predominantly $\beta$-sheet which exists in an oligomeric form in aqueous solution. (Abstract shortened by UMI.)

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Recommended Citation

Fu, Fen-Ni, "Structure and function relationship of botulinum neurotoxin and a neurotoxin associated protein" (1997). Doctoral Dissertations Available from Proquest. AAI9809333.