Computational Design of Unnatural Amino Acid Dependent Metalloproteins

非天然氨基酸依赖性金属蛋白的计算设计

• 批准号: 8391786
• 负责人: Jeremy Mills
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-11-15 至 2013-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391786
• 关键词: Active Sites; Affinity; Alanine; Algorithms; Amino Acids; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Sciences; Bioremediations; Biphenyl Compounds; Bipyridyl; Carbonates; Catechols; Cell Nucleus; Characteristics; Chemicals; Complex; Computer Simulation; Computing Methodologies; Data; Data Coordinating Center; Deposition; Dioxygenases; Dopamine; Engineering; Environment; Enzymes; Epitopes; Excision; Future; Glean; Goals; In Transferrin; Iron; Iron-Binding Proteins; Laboratories; Ligand Binding; Ligands; Metal Binding Site; Metalloproteins; Metals; Methods; Neurotransmitters; Oxygen; Polychlorinated Biphenyls; Process; Protein Analysis; Protein Engineering; Proteins; Reaction; Research; Research Institute; Scaffolding Protein; Side; Site; Structure; System; Techniques; Technology; Therapeutic; Toxic Environmental Substances; Toxin; Universities; Washington; base; cofactor; design; directed evolution; extradiol dioxygenase; functional group; member; metalloenzyme; novel; protein folding; rapid growth; screening; sensor; small molecule; software development; success

DESCRIPTION (provided by applicant): The confluence of the fields of site-specific incorporation of unnatural amino acids and computational protein design represents a currently unexplored but promising avenue of biochemical research. While computational methods have been developed for naturally occurring proteins, the ability to treat non-natural amino acids with these techniques has yet to be fully explored. The research proposed seeks to develop a computational method that allows design of proteins containing unnatural amino acids, with the ultimate goal of generating novel unnatural amino acid dependent enzymes with therapeutic potential. The Rosetta suite of software developed by members of the Baker lab at the University of Washington will first be used to design iron binding proteins that utilize the metal binding unnatural amino acid bipyridyl alanine - first incorporated into proteins by Schultz and co-workers at The Scripps Research Institute. As this unnatural amino acid has inherent affinity for iron, the difficult problem of designing a metal binding site within a protein should be rendered more computationally tractable. As a second goal, a binding site for dopamine (which will provide two oxygen ligands for the iron) will be concurrently engineered. Catechols like dopamine have inherently high affinities for iron suggesting the engineered proteins could serve as sensors for this important class of small molecules. Finally, the catechol binding proteins will be further designed computationally with the goal of creating a non-natural amino acid dependent extradiol dioxygenase like enzyme. Such an enzyme could have a far-reaching impact with respect to bioremediation of persistent anthropomorphic toxins such as polychlorinated biphenyl compounds. The designed unnatural amino acid containing proteins will be produced in a bacterial expression system using techniques developed by members of the Schultz laboratory. Purified proteins will then be analyzed using a host of bioanalytical techniques that will examine metal or catechol binding abilities, or enzymatic activity depending on the specific aim. Data collected in the course of experimentation will be used for future design of other unnatural amino acid containing proteins both within the scope of this project, and beyond. Consequently, this research should have far reaching impacts within the biological sciences that will extend beyond the projects described above. As both of the scientific fields explored in this proposal are currently in a state of rapid growth, any information gleaned in the course of this research will guide further computational design efforts involving other currently available, genetically encoded non-natural amino acids, as well as those developed in the future.

描述（由申请人提供）：不自然氨基酸和计算蛋白设计的现场特异性结合域的汇合代表了目前尚未开发但有希望的生化研究途径。尽管已经开发了用于自然发生的蛋白质的计算方法，但使用这些技术治疗非天然氨基酸的能力尚未得到充分探索。该研究旨在开发一种计算方法，该方法允许设计含有非天然氨基酸的蛋白质，其最终目的是产生具有治疗潜力的新型非天然氨基酸依赖性酶。 华盛顿大学贝克实验室成员开发的软件套件将首先用于设计铁结合蛋白，这些蛋白质利用金属结合的非天然氨基酸双吡啶基丙氨酸 - 首先由Schultz和Scripps Research Institute的同事掺入蛋白质。由于这种不自然的氨基酸对铁具有固有的亲和力，因此在蛋白质中设计金属结合位点的困难问题应在计算方面更具处理性。作为第二个目标，将同时设计多巴胺的结合位点（将为铁提供两个氧配体）。诸如多巴胺之类的儿茶酚具有固有的高亲和力，这表明工程蛋白可以作为这类重要的小分子的传感器。最后，儿茶酚结合蛋白将在计算上进一步设计，目的是创建依赖于酶的非天然氨基酸依赖性氨基酸二氧酶。这种酶在持续的拟人化毒素（如多氯联苯化合物）的生物修复方面可能会产生深远的影响。 设计的非天然氨基酸含有蛋白质将使用由Schultz实验室成员开发的技术在细菌表达系统中产生。然后，将使用许多将检查金属或儿茶酚结合能力的生物分析技术或根据特定目的来检查金属或儿茶酚结合能力的纯化蛋白质。在实验过程中收集的数据将用于将未来的其他不自然氨基酸设计，这些氨基酸含有该项目范围内的其他不自然的氨基酸。 因此，这项研究应该在生物科学中产生远远超出上述项目的影响。正如本提案中探索的两个科学领域目前处于快速增长的状态一样，这项研究过程中收集的任何信息都将指导进一步的计算设计工作，涉及其他当前可用的，基因编码的非天然氨基酸以及未来开发的信息。

项目成果

Transition states. Trapping a transition state in a computationally designed protein bottle.

• DOI: 10.1126/science.aaa2424
• 发表时间: 2015-02-20
• 期刊: Science (New York, N.Y.)
• 作者: Pearson AD;Mills JH;Song Y;Nasertorabi F;Han GW;Baker D;Stevens RC;Schultz PG
• 通讯作者: Schultz PG