MOLECULAR STRUCTURE OF THE 900 KD BOTULINUM NEUROTOXIN COMPLEX

A型肉毒神经毒素复合体是一种900 kDa的蛋白质 由厌氧细菌产生的八种血清型(A-G)之一 肉毒梭菌。已知的最有效的生物毒素之一 人，肉毒杆菌神经毒素导致突触囊泡释放抑制 神经肌肉连接导致迟缓性瘫痪，最终 死亡。A型肉毒神经毒素(BoNT/A)是一种有效的致病因子 食源性肉毒杆菌中毒和婴儿猝死综合征 建立了生物武器，并提出了一种新的治疗方法 不由自主的肌肉紊乱。此前，我们已经确定了3-D 900 kDa复合体150 kDNA神经毒素组分的结构 X射线结晶学。我们还完成了抗体图谱实验 确定150 kDa神经毒素如何结合到900 kDa毒素中 很复杂。我们已经进行了一系列的生物物理稳定性实验 为了了解两个组件(150 kDa毒素和750 kDa)是如何 无毒成分)结合并稳定900 kDa的复合体。最后， 基于以上工作和初步的电子显微镜工作，我们是 设计肉毒杆菌中毒的替代疫苗策略。目前的疫苗 肉毒杆菌中毒的治疗方案并不是很有效。 这项建议的初步目标是获得三个- 900 kDa肉毒神经毒素复合体的空间结构，以及 了解神经毒素成分是如何融入复合体的。至 为了实现这一目标，我们将使用一个900 kDa的二维晶体复合体 进行三维图像重建实验。我们已经得到了2-D 900 kDa晶体复合体进行三维图像重建 实验。我们已经获得了900 kDa复合体的二维晶体 它在负应变下的衍射率弱到14埃分辨率，并且a 制作了30埃分辨率的密度投影图。基座 关于晶体质量和观察到缺陷的频率 在我们早期调查中使用的晶体中，似乎 可以得到质量高得多的晶体。具体地说，我们的转移 由于我们在这一领域的新冒险，目前的技术是粗糙的 研究，并对其他项目提出了几点建议 关于如何改进我们的转移技术的成员。我们也是 研究有助于稳定蛋白质的替代缓冲条件 再远一点。一旦2-D晶体的优化完成，我们将 在可能的最大分辨率下完成负片染色工作 在倾斜序列中收集数据，然后进行三维图像重建。 这项工作之后将尝试更高分辨率的研究 冷冻技术。我们将在在场的情况下结晶900 kDa的复合体 对暴露区域具有高亲和力的单链抗体分子 当与900 kdN复合体结合时，神经毒素的含量。

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.