BORDETELLA CYCLASE--STRUCTURE AND BIOLOGICAL ACTIVITIES

描述：腺苷环化酶(AC)毒素是一种新的蛋白细菌 百日咳病原--百日咳杆菌产生的毒素 咳嗽。这种毒素序列是已知的，且单个毒素的裂解 赖氨酸残留物已被证明是毒素活性所必需的。 然而，对毒素介导的机制知之甚少。 进程或它们的功能相互关系。 这项提案将侧重于AC毒素所涉及的各个步骤 行动。将调整或开发化验方法，以使分离和 对路径中的每一步进行表征。方法论 这些方法将包括使用固有的色氨酸荧光 荧光能量转移、脂质双层和全细胞膜片钳 电导、电子显微镜和x射线衍射仪。功能界别 研究将与随机和特定地点的使用相补充 诱变产生毒素缺陷的AC毒素突变体 和/或溶血素活性。类似地，体外酰化反应将 提供了一种除棕榈酰基以外的制备毒素的机制 LYs983上的基团，以确定酰化作用。其他内容 结构数据将用CD光谱学和x射线衍射法收集 研究以更好地了解AC毒素的作用。

DESCRIPTION: Adenylate cyclase (AC) toxin is a novel protein bacterial toxin produced by Bordetalla pertussis, the causative agent of whooping cough. The sequence of this toxin is known and palimiolyation of a single Lys residue has been demonstrated to be essential for toxin activities. Nevertheless, little is known about mechanisms for toxin-mediated processes or their functional interrelationships. This proposal will focus on the individual steps involved in AC toxin action. Assays will be adapted or developed to enable the isolation and characterization of each individual step in the pathway. The methodologic approaches will include use of intrinsic tryptophan fluorescence fluoresecence energy transfer, lipid bilayer and whole cell patch-clamp conductance, electron microscopy and x-ray diffraction. The functional studies will be complemented with th use of random and site-specific mutagenesis to generate mutants of AC toxin that are defective in toxin and/or hemolysin activities. Similarly, an in vitro acylation will provide a mechanism for preparation of toxins with other than palmitoyl groups on LYs983, in order to determine the role of acylation. Additional structural data will be collected with CD spectroscopy an x-ray diffraction studies to develop a better understanding of the actions of AC toxin.