STRUCTURAL STUDIES ON PROTEINS THAT CONTROL APOPTOSIS, SULFUR ASSIMILATION & NEU

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。SSRL需要时间对来自三种不同生物系统的蛋白质进行高分辨率结构测定。一个系统涉及抑制细胞凋亡的病毒蛋白。在本次提案更新中，我的实验室正在积极寻求确定病毒蛋白与它们所抑制的细胞半胱天冬酶之间复合物的结构。我们最近培育出了解理前和解理后复合物的晶体，在9-1束线上衍射分辨率分别为5°和4.6°。我们目前正在进行实验，以增加晶体的顺序，以改善衍射。第二个系统涉及蛋白质，这些蛋白质将环境中的无机硫吸收到生物体中。我们最近从两个不同的物种中确定了ATP硫化酶的结构，它催化了硫同化的第一步。我们现在将采用位点定向诱变来创建突变体，以研究结构-功能关系，了解变构调节以及从人类研究这些酶。最后一次被要求使用的系统是PLP依赖性酶？-aminobutyrate转氨酶。这种酶是用于治疗癫痫、亨廷顿氏病、精神分裂症和阿尔茨海默病的神经活性药物的重要靶点。我们已经解决了大肠杆菌的酶结构，并将研究与药物和抑制剂配合抑制的结构方面。来自SSRL的高通量将导致更高分辨率的高质量数据，并能够更好地更详细地了解这三种体系中的所有蛋白质结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Time is requested at SSRL for high-resolution structure determination of proteins from three different biological systems. One system involves viral proteins that inhibit apoptosis. In this proposal renewal, my laboratory is actively pursuing structure determination of complexes between the viral protein and the cellular caspases, which they inhibit. We recently have grown crystals both of the pre- and post-cleavage complex that diffract to 5¿¿and 4.6¿¿resolution respectively on beam line 9-1. We are currently pursuing experiments to increase the order in the crystals to improve diffraction. The second system involves proteins that assimilate inorganic sulfur from the environment into living organisms. We have recently determined the structure of the enzyme ATP sulfurylase, which catalyzes the first committed step in sulfur assimilation, from two different species. We will now be employing site-directed mutagenesis to create mutants to study structure-function relationships to understand the allosteric regulation as well as study these enzymes from human. The last system for which time is requested is on a PLP dependent enzyme ?-aminobutyrate aminotransferase. This enzyme is an important target for neuroactive drugs used in treatment of epilepsy, Huntington's disease, schizophrenia and Alzheimer's disease. We have solved the E. coli enzyme structure and will study structural aspects of inhibition complexed with drugs and inhibitors. The high flux from SSRL will result in higher quality data at much higher resolution and enable better more detailed understanding for all the protein structures in these three systems.