MOLECULAR STRUCTURE OF THE 900 KD BOTULINUM NEUROTOXIN COMPLEX

900 KD 肉毒神经毒素复合物的分子结构

• 批准号: 6325880
• 负责人: RAYMOND C STEVENS
• 金额: $ 22.3万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325880
• 关键词: X ray crystallography; botulinum toxins; electron crystallography; electron density; hemagglutinin; neurotoxins; protein structure

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.

肉毒杆菌神经毒素复合物血清型A是一种900千道尔顿（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。已知最强的生物毒素之一， 人，肉毒杆菌神经毒素导致抑制突触囊泡释放， 导致弛缓性麻痹，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症（SIDS）， 建立的生物武器，和一种新的治疗方法， 不随意肌紊乱此前，我们已经确定了三维 900 kDa复合物的150 kDNA神经毒素组分的结构， X射线晶体学我们还完成了抗体定位实验 以确定150 kDa神经毒素如何结合到900 kDa毒素中 复杂.我们进行了一系列生物物理稳定性实验 为了理解两种组装体（150 kDa毒素和750 kDa毒素）如何 无毒组分）联合收割机并稳定900 kDa复合物。最后， 基于上述工作和初步的电子显微镜工作，我们 设计肉毒杆菌中毒的替代疫苗策略。当前疫苗 肉毒杆菌中毒的治疗不是很有效。 该提案的初步目标是获得三个- 900 kDa肉毒杆菌神经毒素复合物的三维结构，和 了解神经毒素成分是如何融入复合体的到 为了实现这一目标，我们将使用900 kDa复合物的2-D晶体， 进行三维图像重建实验。我们已经获得了2D 900 kDa复合物的晶体进行3-D图像重建 实验我们已经获得了900 kDa复合物的二维晶体 在负应变下，该膜对14埃分辨率作用较弱， 密度投影图的分辨率为30埃。基于 晶体质量和观察到缺陷的频率 在我们早期研究中使用的晶体中， 可以获得高得多的质量晶体。具体来说，我们的转移 技术是目前原油，由于我们的新企业进入这一领域， 研究，并提出了一些建议，由其他计划项目 如何改进我们的转让技术。我们也 研究替代缓冲条件以帮助稳定蛋白质 进一步.一旦优化的2-D晶体已经完成，我们将 以最大分辨率完成负染色工作， 在倾斜系列中收集数据，然后进行3-D图像重建。 这项工作之后将尝试更高分辨率的研究， 冷冻技术我们将使900 kDa的复合物在 对暴露区域具有高亲和力的scFv抗体分子 神经毒素结合到900 kDn复合物上的时候。