Site Specific Protein Labeling In Vivo Through Genetically Engineered Phosphopant

通过基因工程磷酸化剂进行体内位点特异性蛋白质标记

• 批准号: 8127733
• 负责人: Michael D. Burkart
• 金额: $ 53.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8127733
• 关键词: 4' -phosphopantetheine; Acyl Carrier Protein; Address; Alanine; Amino Acids; Amino Acyl-tRNA Synthetases; Arsenates; Biochemistry; Biological Process; C-terminal; Carrier Proteins; Cell Culture System; Cell Culture Techniques; Cell Nucleus; Cell surface; Cells; Cellular biology; Chemicals; Chemistry; Coenzyme A; Collection; Complement; Complex; Coupled; Coupling; Cysteine; DNA; Development; Engineering; Environment; Enzymes; Enzymes and Coenzymes; Event; Fatty Acids; Fluorescence Resonance Energy Transfer; Foundations; Funding; Gene Activation; Genetic; Genetic Transcription; Goals; Image; Imagery; In Vitro; Individual; Label; Libraries; Life; Ligands; Mammalian Cell; Metabolic Pathway; Methodology; Molecular Biology; Movement; Mutagenesis; N-terminal; Organism; Pantetheine; Pantothenate kinase; Pathway interactions; Peptides; Post-Translational Protein Processing; Problem Solving; Process; Production; Prokaryotic Cells; Property; Protein Dynamics; Protein Export Pathway; Protein Import; Proteins; Reaction; Reagent; Regulation; Reporting; Research; Resolution; Role; Scheme; Series; Side; Signal Transduction; Site; Structure; System; Techniques; Technology; Tertiary Protein Structure; Time; TimeLine; Variant; Work; Yin; analog; base; directed evolution; enzyme biosynthesis; fluorophore; genetic regulatory protein; human disease; in vivo; member; mutant; novel; pantothenate; phosphopantetheinyl transferase; protein function; public health relevance; response; single molecule; single-molecule FRET; structural biology; tool

DESCRIPTION (provided by applicant): Of fundamental importance to bridging the gap between biochemistry and cell biology is the ability to study proteins in a living cell. A great deal of progress has been made in this regard both on the chemistry side and the imaging side. Many new chemical tools are now in place to visualize proteins in vivo as well as single molecule and ensemble visualization techniques to utilize these tools. However, to date no general and broadly applicable tool exists to label individual proteins in a specific and non-perturbing manner. Moreover, the existing labels do not possess the required photophysical properties for detailed probing of protein dynamics, localization, and interaction with other members of the cellular environment. Current shortfalls of existing techniques include large fluorophores, toxic reagents, cell impermeability, slow maturation times, or poor photophysical properties. A general protein labeling tool that addresses each of these problems and allows incorporation of multiple labels for fluorescence resonance energy transfer or spin-spin coupling studies would provide an invaluable tool to bridge the gap between biochemistry, genetics, and cell biology. One common protein labeling strategy uses an organism's biosynthetic machinery to insert the desired probe. We have previously demonstrated the use of fatty acid biosynthetic machinery in vivo to incorporate non-canonical phosphopantetheine analogues onto truncated modular carrier proteins. This methodology has provided a general labeling scheme, but to date the inefficiency of this system, caused by competition from the natural ligand, coenzyme A (CoA), has prohibited general application. In order to create a robust tool, we will investigate a series of unnatural pantothenate analogues that are not recognized by the first and last enzymes of this biosynthetic pathway. We will follow with directed evolution of these two enzymes to accept a set of non-canonical substrates in order to provide a protein labeling paradigm that will be fully orthogonal to endogenous machinery. These tools will allow specific and efficient incorporation of desired labels without competing side reactions. After implementation in a mammalian cell culture, we will probe the role of I:B to strip NF:B from DNA in the nucleus of living cells through ensemble and single-molecule FRET techniques. Public Health Relevance: The proposed research will add a general tool to probe protein function in vivo. All organisms require properly functioning and correctly localized gene products to survive, and following the production, movement, and interactivity of gene products remains an important goal for the study of human diseases caused by improper protein import and export, incorrect folding to a functional tertiary structure, and formation of deleterious higher order aggregates or structures. Here we propose the study of a novel protein labeling system that harnesses an existing metabolic pathway to attach a chemical probe onto proteins within living cells. Selective application of these probes will allow us to study important protein activity in living cells, and here we propose to use these tools in the study of gene activation by regulatory proteins.

描述（由申请人提供）：弥合生物化学和细胞生物学之间差距的基本重要性是研究活细胞中蛋白质的能力。在化学方面和成像方面，这方面都取得了很大的进步。现在已经建立了许多新的化学工具，可以在体内可视化蛋白质以及单分子和集合可视化技术来利用这些工具。但是，迄今为止，尚无一般且广泛适用的工具可以以特定和非扰动方式标记单个蛋白质。此外，现有标签不具有所需的光物理特性，用于详细探测蛋白质动力学，定位以及与细胞环境其他成员的相互作用。现有技术的当前缺口包括大型荧光团，有毒试剂，细胞不渗光，成熟时间缓慢或光物理特性差。一种解决这些问题的通用蛋白质标记工具，并允许将多个标签纳入用于荧光共振能量转移或自旋旋转耦合研究的多个标签，这将为弥合生物化学，遗传学和细胞生物学之间的差距的宝贵工具。一种常见的蛋白质标记策略使用生物体的生物合成机械插入所需的探针。我们先前已经证明了在体内使用脂肪酸生物合成机械的使用，将非典型的磷酸耐氨基类似物掺入截短的模块化载体蛋白上。该方法提供了一般的标签方案，但迄今为止，由于天然配体辅酶A（COA）竞争引起的该系统的效率低下，已禁止一般应用。为了创建一个健壮的工具，我们将研究一系列不自然的泛素类似物，这些类似物被该生物合成途径的第一个也是最后一个酶所识别。我们将遵循这两种酶的定向演变，以接受一组非典型底物，以提供蛋白质标记范式，该范围将完全正交到内源机械。这些工具将允许在不竞争侧面反应的情况下特定，有效地掺入所需的标签。在哺乳动物细胞培养物中实施后，我们将通过集合和单分子FRET技术探测I：B到Live细胞核中DNA的作用。公共卫生相关性：拟议的研究将为体内探测蛋白质功能的一般工具。所有生物都需要正常运行并正确局部的基因产物才能生存，并且基因产物的生产，运动和相互作用仍然是研究由不当蛋白质进口和出口引起的人类疾病的重要目标，不正确地折叠到功能性的第三纪结构，并形成了可恶的高级聚集物或结构。在这里，我们提出了一种新型蛋白质标记系统的研究，该系统利用现有的代谢途径将化学探针连接到活细胞内的蛋白质上。这些探针的选择性应用将使我们能够研究活细胞中的重要蛋白活性，在这里我们建议在调节蛋白研究基因激活中使用这些工具。

项目成果

The complexities of p97 function in health and disease.

• DOI: 10.1039/c0mb00176g
• 发表时间: 2011-03
• 期刊: Molecular bioSystems
• 作者: Chapman E;Fry AN;Kang M
• 通讯作者: Kang M

Structure of FD-895 revealed through total synthesis.

通过全合成揭示了 FD-895 的结构。

• DOI: 10.1021/ol3023006
• 发表时间: 2012
• 期刊: Organic letters
• 影响因子: 5.2
• 作者: Villa,Reymundo;Mandel,AlexanderL;Jones,BrianD;LaClair,JamesJ;Burkart,MichaelD
• 通讯作者: Burkart,MichaelD