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.

项目摘要 将非典型氨基酸（ncAA）基因整合到位点特异性氨基酸链中的能力是一种基因工程。 通过提供新的研究工具， 和工程蛋白质，并在几个生物医学领域产生了显着的影响 包括调节蛋白质功能，蛋白质的体内成像，以及设计新的 治疗学在该技术中，正交氨酰-tRNA合成酶和tRNA对 (RS/tRNA）被添加到细胞中。尽管有这些优势 由ncAA公司提供，RS进化的库大小的实际限制 限制在每轮可以突变的残基的数目。因此，一些有益的 忽略了第一壳层中的相互作用和所有第二壳层的相互作用。因此，我们认为， 选择的ncAA-RS与野生型翻译的催化常数不匹配，导致低翻译活性。 ncAA-蛋白表达产率和在许多蛋白表达条件下缺乏选择性。 罗塞塔计算设计程序提供了一个令人兴奋的新的选择，克服这一关键 限制遗传密码的扩展。基于四嗪的氨基酸（Tet-ncAA）提供了极其 用于蛋白质的位点特异性标记的快速和稳健的生物正交化学，因此将是 基于Rosetta的优化的理想模型系统。快速蛋白质生物正交连接是 正在生物医学研究和材料科学中实施，用于许多应用， 体内成像、探测蛋白质功能、药物递送和蛋白质-聚合物杂化物。 除了上述原因外，工程Tet-ncAA-RS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。 在这个建议中，我将使用Rosetta来设计更好的Tet-RS，并获得结构上的见解， 这些残基对结合很重要。这些信息指导着“智能图书馆”的生成 通过筛选较少的变体以克服大小限制，具有更高的成功机会。在 同时，我还将通过设计新的协议来改进Rosetta的现有功能 以及增强分数函数。这些增强和补充将公开提供。 用于高效和选择性掺入Tet-ncAA的RS的上级集合的设计 为科学家提供了一种理想的工具，用于在体内快速特异性标记蛋白质， 生物正交法这种能力对于许多应用是明确的重要性， 生物医学研究建议的策略可以推广到其他ncAA或解决 遗传密码扩展领域的其他问题。它还将为一个持久的 计算蛋白质设计和遗传密码扩展领域之间的合作。