Studies on the Interaction between Subunits of the Botulinum Toxin Complex and X-Ray Crystallography

肉毒杆菌毒素是一种650 kDa的毒素复合物（TC），其亚基结构与五种不同的蛋白质成分，150 kDa的神经毒素（NT），无毒的非血凝素（NTNHA）和三种血凝素亚组分在培养上清液中非共价结合。在本研究中，我们明确了TC中亚基组分的分子比例。D型菌株4947（D-4947）产生大量TC （650 kDa L-TC、610 kDa TC和540 kDa TC）。它们在SDS-PAGE上显示相同的带型，但在TC上的HA-33/17复合物数量较少。通过HA-33/17配合物的滴定实验，我们得出TC的完整亚基组成是由1个NT、1个NTNHA、2个HA-70、4个HA-33和4个HA-17分子组成的十二聚体。因此，我们对用x射线晶体学分析肉毒杆菌TC的三维结构有了初步的了解。在TC物种中，根据温度等储存条件，在610和540 kDa的TC中发现了组分的重组，因此通过添加甘油来抑制实验中稳定TC的供应。此外，我们建立了新的方法，从D-4947 TC中分离出大量的NT，这在技术上是很难分离的。虽然我们已经尝试通过130种不同的实验条件组合来结晶C和D血清型菌株的动态光散射检测的tc (M和L-TC)，但尚未获得用于x射线分析的大晶体。

Botulinum toxin is produced as a 650 kDa toxin complex(TC) with subunit structure that non-covalently associated with five different protein components, 150 kDa neurotoxin(NT), nontoxic non-hemagglutinin(NTNHA) and three hemagglutinin subcomponents, in culture supernatant. In this study, we have clarified molecular ration of subunit components in the TC. Type D strain 4947(D-4947) produces a large amount of TC species (650 kDa L-TC,610 kDa and 540 kDa TC). They revealed same banding patterns on SDS-PAGE, but less number of HA-33/17 complexes in the TC. Based on titration experiments with HA-33/17 complex, we have concluded that complete subunit composition of the TC is a dodecamer composed of a single NT, a single NTNHA, two HA-70,four HA-33 and four HA-17 molecules. Accordingly, we have preliminary information on the analysis of the three-dimensional structure of botulinum TC by X-ray crystallography. Of the TC species, recombination of the components was found in 610 and 540 kDa TCs depending upon storage condition such as temperature, and was thus depressed by the addition of glycerol leading supply of stable TC for the experiments. Additionally, we have established new method to separate considerable amount of the NT from the D-4947 TC, which has been technically difficult to separate. Although we have attempted to crystallize TCs (M and L-TC) that were examined using dynamic light scattering from serotype C and D strains by 130 combinations of experimental condition, it has not been yet obtain large crystals for X-ray analysis.