MOLECULAR MECHANISMS OF BACTERIAL PATHOGENESIS

这个项目的长期目标是了解结构- ADP-核糖基化外毒素的功能关系 三维晶体结构的水平。 工作 描述的是集中在白喉毒素（DT）和假单胞菌 铜绿外毒素A（ETA），两种相关的毒素， 通过延伸因子-2（EF-2）的ADP-核糖基化的蛋白质合成 2）。 关于DT，将进行以下研究： 通过结合位点定向的 用各种生物化学和生物物理方法诱变; (ii)通过以下方法表征片段A与EF-2的相互作用： 新的方法，包括使用由 有机化学和新的检测方法， 具有活性毒素片段的EF-2;（iii）选择毒素突变体 在膜插入和/或通道方面有缺陷的细胞 在酸性pH下通过新的正选择形成 方法在E.（iv）探索一种新的研究方法 DT的酶活性片段A部分的移位 至平面脂质双层的反面;和（v）确定 3-三维结构的单体天然DT，一个酶 活性片段（片段A）和特定的突变形式 兴趣 关于ETA，建议进行的研究主要 与上述DT相似。 将对结果进行解读 根据测定的ETA的3.0 A晶体结构 最近，和一个新兴的三维结构的DT-二聚体在 类似决议。 这些研究与各种 生物医学科学中的问题，包括 蛋白质穿透细胞膜，成为新疫苗的途径， 将有毒蛋白导向特定细胞的机制。

The long range goals of this project are to understand structure- function relationships of the ADP -ribosylating exotoxins at the level of 3-dimensional crystallographic structure. The work described is focused on diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA), two related toxins that block protein synthesis by ADP-ribosylation of Elongation Factor-2 (EF- 2). With respect to DT, studies will be performed: (i) to characterize the active site, by combining site-directed mutagenesis with various biochemical and biophysical methods; (ii) to characterize the interaction of fragment A with EF-2 by new approaches, including the use of diphthamide synthesized by organic chemistry, and new assays for detecting interaction of EF-2 with active toxin fragments; (iii) to select toxin mutants that are defective in membrane-insertion and/or channel- formation at acidic pH, by means of a new positive selection procedure in E. coli; (iv) to explore a new method to study translocation of the enzymically active fragment A moiety of DT to the trans face of planar lipid bilayers; and (v) to determine the 3-dimensional structures of monomeric native DT, an enzymically active fragment (fragment A), and mutant forms of particular interest. With respect to ETA, studies are proposed which largely parallel those describe above for DT. Results will be interpreted in terms of a 3.0 A crystallographic structure of ETA determined recently, and an emerging 3-dimensional structure of DT-dimer at similar resolution. These studies are pertinent to a variety of problems in biomedical science, including mechanisms by which proteins penetrate membranes, routes to new vaccines, and mechanisms of directing toxic proteins to specific cells.