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: 7556737
• 负责人: Michael D. Burkart
• 金额: $ 57.83万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7556737
• 关键词: 4' -phosphopantetheine; Acyl Carrier Protein; Address; Alanine; Amino Acids; Amino Acyl-tRNA Synthetases; Arsenates; Arts; Biochemistry; Biological Process; C-terminal; Carrier Proteins; Cell Culture System; 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; Localized; 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; Prohibit; Prokaryotic Cells; Property; Protein Dynamics; Protein Import; Proteins; Public Health; 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; desire; directed evolution; enzyme biosynthesis; fluorophore; genetic regulatory protein; human disease; in vivo; intracellular protein transport; member; mutant; novel; pantothenate; phosphopantetheinyl transferase; protein function; protein localization location; response; single molecule; single-molecule FRET; size; 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）：对于弥合生物化学和细胞生物学之间的差距至关重要的是研究活细胞中蛋白质的能力。在这方面，无论是化学方面还是成像方面都取得了很大的进展。现在已经有了许多新的化学工具来可视化体内蛋白质，以及利用这些工具的单分子和整体可视化技术。然而，迄今为止，还没有通用且广泛适用的工具来以特定且无干扰的方式标记单个蛋白质。此外，现有的标记不具备详细探测蛋白质动力学、定位以及与细胞环境其他成员相互作用所需的光物理特性。目前现有技术的缺点包括荧光团大、试剂有毒、细胞不渗透性、成熟时间慢或光物理性质差。通用的蛋白质标记工具可以解决上述每个问题，并允许结合多个标记进行荧光共振能量转移或自旋-自旋耦合研究，这将为弥合生物化学、遗传学和细胞生物学之间的差距提供宝贵的工具。一种常见的蛋白质标记策略使用生物体的生物合成机制来插入所需的探针。我们之前已经证明了体内脂肪酸生物合成机制的使用，将非经典磷酸泛硫氨酸类似物整合到截短的模块化载体蛋白上。该方法提供了通用的标记方案，但迄今为止，由于天然配体辅酶 A (CoA) 的竞争，该系统效率低下，阻碍了普遍应用。为了创建一个强大的工具，我们将研究一系列不被该生物合成途径的第一个和最后一个酶识别的非天然泛酸类似物。我们将跟踪这两种酶的定向进化，以接受一组非规范底物，以提供与内源机制完全正交的蛋白质标记范例。这些工具将允许特定且有效地掺入所需标签，而不会产生竞争性副反应。在哺乳动物细胞培养中实施后，我们将通过集成和单分子 FRET 技术探讨 I:B 从活细胞核中 DNA 中剥离 NF:B 的作用。公共健康相关性：拟议的研究将添加一种通用工具来探测体内蛋白质功能。所有生物体都需要正常运作和正确定位的基因产物才能生存，并且跟踪基因产物的产生、运动和相互作用仍然是研究因蛋白质进出口不当、功能性三级结构不正确折叠而引起的人类疾病的重要目标，并形成有害的高阶聚集体或结构。在这里，我们建议研究一种新型蛋白质标记系统，该系统利用现有的代谢途径将化学探针附着到活细胞内的蛋白质上。选择性应用这些探针将使我们能够研究活细胞中重要的蛋白质活性，在这里我们建议使用这些工具来研究调节蛋白的基因激活。