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

项目摘要 在基因上将非规范氨基酸(NCAA)整合到特定部位的能力 方式为蛋白质生物化学领域带来了革命性的变化，为研究提供了新的工具 和工程蛋白质，并在几个生物医学领域产生了显著的影响 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。在这项技术中，正交氨酰基-tRNA合成酶和tRNA对 为新的NCAA结构进化的(RS/tRNA)被添加到细胞中。尽管有这些优势 由NCAA公司提供，RS进化的文库大小的实际限制 限制每一轮可以突变的残基数量。因此，几个有益的 第一个壳中的交互作用和所有第二个壳交互作用被忽略。因此， 选定的NCAA-RSS与野生型翻译的催化常数不匹配，导致低 NCAA-在许多蛋白质表达条件下，蛋白质表达的产率和缺乏选择性。 Rosetta计算设计程序为克服这一关键问题提供了一个令人兴奋的新选择 遗传密码扩展方面的限制。基于四嗪的氨基酸(Tet-NCAA)提供了极高的 快速和强大的生物正交化学用于蛋白质的位点特异性标记，因此将是 一个理想的基于Rosetta的优化模型系统。快速蛋白质生物正交连接是 在生物医学研究和材料科学中实现了许多应用，包括 活体成像、探测蛋白质功能、药物输送和蛋白质-聚合物杂化。 除上述原因外，工程TET-NCAA-RSS具有独特的挑战性 因为活性更强的Tet-NCAA给选择方法增加了额外的压力。 在这个提案中，我将使用Rosetta来设计更好的Tet-RSS，并获得对 对结合很重要的残基。这些信息指导着“智慧图书馆”的产生 通过筛选较少的变异体来克服大小限制，成功的机会更高。在……里面 同时，我还将通过设计新的协议来改进Rosetta中的现有功能 增强了评分功能。这一增强和增加将公开提供。 高效选择性掺入TET-NCAAs的RSS优选集设计 为科学家提供了一种理想的工具，用于快速和快速地在体内对蛋白质进行定点标记 生物正交法。这一能力对于许多应用程序来说都具有明确的重要性 生物医学研究。建议的战略可以推广到其他非国家评估机构或解决 遗传密码扩展领域的其他问题。它还将为持久的 计算蛋白质设计和遗传密码扩展领域之间的合作。

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.