MOLECULAR BIOLOGY AND RECOMBINANT PROTEIN

将建立与寄生虫病有关的木瓜蛋白酶家族半胱氨酸蛋白酶的重组表达系统。特别是原生动物和蠕虫的半胱氨酸蛋白酶，包括克氏锥虫、锥虫。布氏杆菌、杜氏利什曼原虫和恶性疟原虫将在大肠杆菌、巴氏杆菌和/或哺乳动物细胞表达系统中表达。这些酶的延伸底物专一性将使用组合方法来确定，这些方法可以确定N端和C端与剪切性多肽键结合的口袋的首选多肽底物。将为这些蛋白酶确定最佳底物，并用于协助该计划项目的高通量筛选工作，以确定抑制剂。此外，特异性图谱信息将与来自相关半胱氨酸蛋白酶的数据相结合，以确定排列在结合口袋中的关键氨基酸，并作为底物特异性的假定决定因素。将进行现场定向替换 这些变异酶被用来测试这些氨基酸在底物识别和酶周转中的作用。利用克鲁扎因作为其他半胱氨酸蛋白酶的战略模板，将制备高度特异的多肽基酰氧甲基酮抑制剂和单链抗体抑制剂，并绘制扩展的底物结合口袋。这些抑制剂将用于利用核磁共振波谱来探测酶在溶液中的活性位置。Cruzain将用13C/15N统一标记和/或标记特定氨基酸。核磁共振将被用来确定当克鲁扎因在溶液中游离时和结合不同的抑制剂时，活性部位和结合口袋中是否存在构象灵活性。活性部位组氨酸也将在核磁共振实验中被监测，该实验测量咪达唑环的耦合常数，以跟踪组氨酸的质子化状态和互变异构状态。将这些变化与抑制剂结合联系起来，将有助于该计划项目的共同努力，开发半胱氨酸蛋白酶抑制剂作为新的抗寄生虫药物。

Recombinant expression systems will be established for papain family cysteine proteases that have been implicated in parasitic diseases. In particular, cysteine proteases of protozoan and helminth parasites including Trypanosoma cruzi, Trypanosoma. brucei, Leishmania donovani and Plasmodium falciparum will be expressed in E. coli, P. pastoris, and/or mammalian cell expression systems. The extended substrate specificity of these enzymes will be determined using combinatorial methods that can identify the preferred peptide substrates for the binding pockets both N-terminal and C-terminal to the scissile peptide bond. Optimal substrates will be identified for the proteases and used to assist in high throughput screening efforts of the program project to identify inhibitors. Furthermore, the specificity profiling information will be combined with data from related cysteine proteases to identify key amino acids that line the binding pocket and serve as putative determinants of substrate specificity. Site directed substitutions will be made and the variant enzymes profiled to test the role of these amino acids in substrate recognition and enzyme turnover. Using cruzain as a strategic template for the other cysteine proteases, peptide-based acyloxymethyl ketone inhibitors and single chain antibody inhibitors will be made that are highly specific and map the extended substrate binding pockets. These inhibitors will be used to probe the active site of the enzyme in solution using NMR spectroscopy. Cruzain will be uniformly labeled and/or labeled at specific amino acids with 13C/15N. NMR will be used to determine if conformational flexibility exists in the active site and binding pockets of cruzain when free in solution and upon binding different inhibitors. The active site histidine will also be monitored in NMR experiments that measure the coupling constants of the imadazole ring to follow the protonation state and tautomeric state of the histidine. Correlating these changes with inhibitor binding will assist in the concerted efforts of the program project to develop cysteine protease inhibitors as new anti-parasitic drugs.