A biophysical characterization of botulinum toxin and cholera toxin

This thesis describes the biophysical characterization of two microbial toxins--botulinum toxin and cholera toxin--(md applications of this characterization to the development of new bio-sensors for these deadly pathogens.;Electron microscopy was used to acquire a two-dimensional image map of the 900 kDa botulinum toxin complex. Based upon the image map, and the hydrodynamic radius, a model for the three-dimensional structure of the complex is presented. The stability of the complex, the toxin, and the non-toxic proteins was examined using circular dichroism and dynamic light scattering under a variety of buffers ranging from pH 2-10. Results indicated that, when uncomplexed, the toxin and non-toxic proteins are least stable at acidic pH; however, when complexed, the proteins are most stable at acidic pH. The stability was further studied by performing protease incubations with pepsin, trypsin, chymotrypsin and carboxypeptidase. Uncomplexed botulinum toxin and the uncomplexed non-toxic proteins were completely proteolyzed. Yet, when in the complexed form, the botulinum toxin survived the protease incubations. Based on these experiments, a molecular rationale for the delivery of botulinum toxin, through the gastrointestinal tract, is proposed. To further the understanding of the relationship between the toxin and the complex, the structural relationship between the 150 kilodalton toxin and the 900 kilodalton complex was investigated by antibody mapping. The mapping used 44 single-chain variable fragments that recognized the individual domains of the toxin. Surprisingly, the results of the mapping indicated that the binding domain, in comparison to the catalytic domain, was covered by the complex. These findings are critical for the development of botulinum toxin vaccines, which target the binding domain.;To further the understanding of toxin-receptor recognition, the cholera toxin-ganglioside system was investigated. The binding series for the ganglioside family was correlated with the structure of the cholera toxin binding domain to determine the most important oligosaccharide residues involved in the cholera toxin-receptor interaction. Finally, the knowledge of bacterial toxin-receptor interactions was combined with recent advances in the field of material science to develop a new type of bio-sensor film that detects the binding of toxins to synthetic membranes and induces a colorimetric response.