Advancing genetic code expansion with Rosetta computational design: improving machinery for bioorthogonal amino acids

通过 Rosetta 计算设计推进遗传密码扩展：改进生物正交氨基酸的机制

• 批准号: 9189236
• 负责人: Parisa Hosseinzadeh
• 金额: $ 5.25万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189236
• 关键词: Acid Fast Bacillae Staining Method; Active Sites; Address; Affinity; Algorithms; Amino Acids; Amino Acyl-tRNA Synthetases; Area; Binding; Biological Models; Biomedical Research; Catalysis; Cells; Chemistry; Collaborations; Computer Simulation; Directed Molecular Evolution; Drug Delivery Systems; Engineering; Evolution; Feedback; Foundations; Future; Generations; Genetic Code; Goals; Health; Hot Spot; Hybrids; Label; Libraries; Ligands; Ligation; Methods; Molecular; Mutate; Mutation; Nature; Operative Surgical Procedures; Polymers; Post-Translational Protein Processing; Protein Biochemistry; Protein Engineering; Proteins; Protocols documentation; Reaction; Research; Scientist; Series; Site; Stress; Structure; Techniques; Technology; Tetanus Helper Peptide; Time; Transfer RNA; Translations; Variant; base; cost; design; fluorescence imaging; functional group; imaging probe; improved; in vivo; in vivo imaging; insight; materials science; novel; novel strategies; novel therapeutics; programs; protein expression; protein function; protein structure; research study; screening; success; tool

Project Summary The ability to genetically incorporate non-canonical amino acids (ncAAs) in a site-specific manner has revolutionized the field of protein biochemistry by providing novel tools for studying and engineering proteins and has had a pronounced influence in several biomedical fields including regulating protein function, in vivo imaging of proteins, and designing novel therapeutics. In this technology an orthogonal amino acyl-tRNA synthetase and tRNA pair (RS/tRNA) that is evolved for new ncAA structure is added to the cell. Despite the advantages offered by ncAA incorporation, the practical limits on the size of the libraries for RS evolution restrict the number of residues that can be mutated at each round. Hence, several beneficial interactions in the first shell and all second shell interactions are overlooked. Therefore, selected ncAA-RSs don't match the catalytic constants of wild type translation resulting in low ncAA-protein expression yield and lack of selectivity under many protein expression conditions. Rosetta computational design program offers an exciting novel option for overcoming this key limitation in genetic code expansion. Tetrazine-based amino acids (Tet-ncAAs) offer extremely fast and robust bioorthogonal chemistry for site specific labeling of proteins and therefore will be an ideal model system for Rosetta based optimization. Fast protein bioorthogonal ligations are being implemented in biomedical research and material science for many applications including in vivo imaging, probing protein function, drug delivery, and protein-polymer hybrids. Engineering Tet-ncAA-RSs is uniquely challenging in addition to the above-mentioned reasons because the more reactive Tet-ncAAs add additional stress to selection methods. In this proposal, I will use Rosetta to design better Tet-RSs and to obtain structural insights into the residues important for binding. This information guides the generation of “smart libraries” with a higher chance of success via screening lesser variants to overcome the size limitation. In parallel, I will also improve upon current functionalities in Rosetta by designing novel protocols and enhancing score functions. This enhancements and additions will be publicly available. The design of superior sets of RSs for efficient and selective incorporation of Tet-ncAAs provides scientists with an ideal tool for site-specific labeling of proteins in vivo in a fast and bioorthogonal manner. This ability is of unequivocal importance for many applications in biomedical research. The proposed strategy can be generalized to other ncAAs or to address other issues in the field of genetic code expansion. It will also lay the foundations of a lasting collaboration between the fields of computational protein design and genetic code expansion.

项目概要 将非规范氨基酸 (ncAA) 基因整合到特定位点的能力 通过提供新的研究工具，彻底改变了蛋白质生物化学领域 和工程蛋白质，并在多个生物医学领域产生了显着影响 包括调节蛋白质功能、蛋白质体内成像以及设计新型蛋白质 在该技术中，正交氨酰-tRNA 合成酶和 tRNA 对。 尽管有这些优点，但将针对新 ncAA 结构进化而来的 (RS/tRNA) 添加到细胞中。 由 ncAA incorporation 提供，RS 进化库大小的实际限制 限制每轮可以突变的残基数量因此，有几个有益的。 因此，第一个 shell 中的交互和所有第二个 shell 交互都被忽略。 选定的 ncAA-RS 与野生型翻译的催化常数不匹配，导致低 ncAA-蛋白质表达产量和在许多蛋白质表达条件下缺乏选择性。 Rosetta 计算设计程序为克服这一关键问题提供了令人兴奋的新颖选择 四嗪基氨基酸（Tet-ncAAs）的限制极大。 快速而强大的生物正交化学用于蛋白质的位点特异性标记，因此将 基于 Rosetta 的优化的理想模型系统是 正在生物医学研究和材料科学的许多应用中实施，包括 体内成像、探测蛋白质功能、药物输送和蛋白质-聚合物杂化物。 除了上述原因外，Tet-ncAA-RS 的工程设计也具有独特的挑战性 因为更具反应性的 Tet-ncAA 给选择方法增加了额外的压力。 在本提案中，我将使用 Rosetta 设计更好的 Tet-RS 并获得结构见解 该信息指导“智能库”的生成。 通过筛选较少的变体来克服尺寸限制，成功的机会更大。 同时，我还将通过设计新颖的协议来改进 Rosetta 中的当前功能 并增强评分功能。此增强功能和添加内容将公开。 设计优质 RS 组，以高效、选择性地掺入 Tet-ncAA 为科学家提供了一种理想的工具，可以快速、准确地对体内蛋白质进行位点特异性标记。 这种能力对于许多应用来说具有明确的重要性。 所提出的策略可以推广到其他 ncAA 或解决问题。 它还将为遗传密码扩展领域的其他问题奠定基础。 计算蛋白质设计和遗传密码扩展领域之间的合作。