Botulinum neurotoxin complex serotype A is a 900 kiloDalton (kDa) protein produced as one of eight serotypes (A-G) by the anaerobic bacterium Clostridium botulinum. Among the most potent biological toxins known to man, botulinum neurotoxin causes inhibition of synaptic vesicle release at the neuromuscular junction resulting in flaccid paralysis and ultimately death. Botulinum neurotoxin type A (BoNT/A) is a potent disease agent in both food-borne botulism and Sudden Infant Death Syndrome (SIDS), an established biological weapon, and a novel therapeutic in the treatment of involuntary muscle disorders. Previously, we have determined the 3-D structure of the 150 kDNA neurotoxin component of the 900 kDa complex by x-ray crystallography. We have also completed antibody mapping experiments to determine how the 150 kDa neurotoxin is bound into the 900 kDa toxin complex. We have conducted a series of biophysical stability experiments in order to understand how the two assemblies (150 kDa toxin and 750 kDa non-toxic component) combine and stabilize the 900 kDa complex. Lastly, based on the work above, and preliminary electron microscopy work, we are designing an alternative vaccine strategy for botulism. Current vaccine programs for botulism are not very effective. The preliminary objective of this proposal is to obtain a three- dimensional structure of the 900 kDa botulinum neurotoxin complex, and understand how the neurotoxin component fits into the complex. To accomplish this goal, we will use a 2-D crystals of the 900 kDa complex to conduct 3-D image reconstruction experiments. We have already obtained 2-D crystals of the 900 kDa complex to conduct 3-D image reconstruction experiments. We have already obtained 2-D crystals of the 900 kDa complex that diffract weakly to 14 Angstroms resolution in negative strain, and a density projection map has been produced at 30 Angstroms resolution. Based on the crystal quality and the frequency with which defects were observed in the crystals used in our earlier investigation, it appears as though much higher quality crystals can be obtained. Specifically, our transfer technique is presently crude due to our new venture into this area of research, and several suggestions have been made by other program project members on how to improve our transfer techniques. We are also investigating alternative buffer conditions to help stabilize the protein further. Once optimization of the 2-D crystals has been completed, we will complete the negative stain work at the maximum resolution possible using data collection in a tilt series followed by 3-D image reconstruction. This work will be followed by attempting higher resolution studies with cryo-techniques. We will crystallize the 900 kDa complex in the presence of scFv antibody molecules that have a high affinity for exposed regions of the neurotoxin when bound to the 900 kDn complex.

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National Institute of General Medical Sciences (NIGMS)
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Lawrence Berkeley National Laboratory
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