Site-specific protein labeling in cells with engineered LplA

描述（由申请人提供）：本提案的目的是开发基于酶硫辛酸连接酶（LplA）的新探针靶向方法。LplA催化辅因子硫辛酸与三种细菌蛋白质底物之一的赖氨酸侧链的位点特异性共价缀合。我们建议重新设计LplA用于活哺乳动物细胞内的探针靶向。我们先前设计了LplA的可转座的12-氨基酸肽底物，并诱变了硫辛酸结合口袋以容纳香豆素荧光团。在这里，我们提出（1）使用合理设计和定向进化的组合来工程化用于红色荧光团试卤灵和光交联剂二苯甲酮的LplA衍生的连接酶。对于定向进化，我们将并行探索FACS，细胞激光消融和抗生素选择策略;（2）开发基于LplA催化的官能团手柄（如烷基叠氮化物）连接的两步探针靶向方案;（3）将我们的新探针连接酶应用于活神经元中neurexin和neuroligin生物学的研究。我们的工作应该有利于对单个活细胞中蛋白质功能的微创分析感兴趣的细胞生物学家，以及对理解突触发育的分子基础感兴趣的神经科学家。 公共卫生相关性：我们正在提出新的化学技术，有可能改变生物显微镜。作为获得今年诺贝尔化学奖的绿色荧光蛋白的替代品，我们的“探针连接酶”技术对所研究的蛋白质的侵入性要小得多，并且可以引入种类更广泛的荧光团、光交联剂和其他探针结构，这些结构具有比GFP发色团更优越的上级特性。在这项提案中，我们还计划使用探针连接酶技术深入研究neurexin和neuroligin生物学;这些研究将揭示突触发育的分子机制，并可能加速发现治疗自闭症等疾病的药物。

DESCRIPTION (provided by applicant): The objective of this proposal is to develop new probe targeting methodology based on the enzyme lipoic acid ligase (LplA). LplA catalyzes the site-specific covalent conjugation of the cofactor lipoic acid onto the lysine sidechain of one of three bacterial protein substrates. We propose to re-engineer LplA for probe targeting inside living mammalian cells. We previously engineered a transposable 12-amino acid peptide substrate for LplA, and mutagenized the lipoate binding pocket to accommodate a coumarin fluorophore. Here, we propose to (1) use a combination of rational design and directed evolution to engineer LplA-derived ligases for the red fluorophore resorufin and the photocrosslinker benzophenone. For directed evolution, we will explore FACS, cell laser ablation, and antibiotic selection strategies in parallel; (2) develop two-step probe targeting schemes based on LplA-catalyzed ligation of functional group handles such as alkyl azides; and (3) apply our new probe ligases to the study of neurexin and neuroligin biology in living neurons. Our work should benefit cell biologists interested in minimally- invasive analysis of protein function in single living cells, and neuroscientists interested in understanding the molecular bases of synapse development. PUBLIC HEALTH RELEVANCE: We are proposing new chemical technology that has the potential to transform biological microscopy. As an alternative to Green Fluorescent Protein, which received this year's Nobel Prize in Chemistry, our "probe ligase" technology is far less invasive to proteins under study, and can introduce a much wider variety of fluorophore, photocrosslinker, and other probe structures with superior properties to the GFP chromophore. In this proposal, we also plan in-depth studies of neurexin and neuroligin biology, using probe ligase technology; these studies will shed light on the molecular mechanisms of synapse development and could accelerate the discovery of drugs to treat diseases such as autism.