New Amino Acids for Protein Engineering

描述（由申请人提供）：该项目将开发强大的新工具来确定复杂细胞系统中蛋白质的制造时间和地点。活性氨基酸类似物将被整合到细胞蛋白中，并选择性地偶联到探针上，用于原核和真核细胞中新合成蛋白的可视化、分离和鉴定。最重要的是，这些方法可以为细胞蛋白质合成分析提供空间和时间分辨率。细胞选择性是通过控制突变氨基酰- trna合成酶的表达来实现的；在包含多种细胞类型的系统中，氨基酸标记仅限于突变合成酶活跃的细胞。该项目将探索使用这些方法来阐明细菌逃避哺乳动物宿主防御的机制，检查群体感应过程（这对细菌病原体表达毒力至关重要），并以细胞选择性的方式询问模式生物秀丽隐杆线虫的蛋白质合成。这些研究将为从发育到疾病治疗的生物现象的系统级表征建立强大的通用平台。

DESCRIPTION (provided by applicant): This project will develop powerful new tools to determine when and where proteins are made in complex cellular systems. Reactive amino acid analogs will be incorporated into cellular proteins and selectively conjugated to probes for visualization, isolation and identification of newly synthesized proteins in both prokaryotic and eukaryotic cells. Most importantly, these approaches can provide both spatial and temporal resolution in analysis of cellular protein synthesis. Cell-selectivity is achieved by controlled expression of mutant aminoacyl-tRNA synthetases; in systems containing multiple cell types, amino acid labeling is confined to cells in which the mutant synthetase is active. This project will explore the use of such methods to elucidate the mechanisms by which bacteria evade the defenses of mammalian hosts, to examine the process of quorum sensing (which is essential to the expression of virulence by bacterial pathogens), and to interrogate protein synthesis in a cell-selective manner in the model organism Caenorhabditis elegans. These studies will establish powerful, general platforms for systems-level characterization of biological phenomena ranging from development to the treatment of disease. PUBLIC HEALTH RELEVANCE: This project will provide new methods to elucidate the mechanisms by which bacteria attempt to evade the defenses of their mammalian hosts, to examine how bacteria communicate with one another to express virulence factors and form antibiotic-resistant biofilms, and to identify the different sets of proteins made by individual cells in living animals. These studies will establish new windows on biological phenomena ranging from growth and development to the treatment of infectious disease.