Site-specific protein labeling in cells with engineered LplA

描述（由申请人提供）：本提案的目标是基于硫辛酸连接酶（LplA）开发新的探针靶向方法。LplA催化硫辛酸辅因子在三种细菌蛋白底物之一的赖氨酸侧链上的位点特异性共价偶联。我们建议重新设计LplA用于探针靶向活的哺乳动物细胞。我们之前为LplA设计了一个转座的12个氨基酸肽底物，并对脂酸结合袋进行了诱变，以容纳香豆素荧光团。在这里，我们提出(1)结合理性设计和定向进化来设计lpla衍生的连接酶，用于红色荧光团间苯二酚和光交联剂二苯甲酮。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)基于lpla催化连接烷基叠氮化物等官能团的两步探针靶向方案；(3)将我们的新型探针连接酶应用于活神经元中神经素和神经素的生物学研究。我们的工作将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，以及对了解突触发育的分子基础感兴趣的神经科学家。

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.