Controlling Protein Post-translational Modification by Separating Affinity and Catalysis in Designer Enzymes

通过分离设计酶的亲和力和催化作用来控制蛋白质翻译后修饰

• 批准号: 2204094
• 负责人: Albert Bowers
• 金额: $ 49.5万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2204094&HistoricalAwards=false
• 关键词: Controlling; Protein; Post; translational; Modification

With the support of the Chemistry of Life Processes (CLP) program in the Chemistry Division, Dr. Albert A. Bowers from the University of North Carolina at Chapel Hill will investigate methods for making artificial enzymes. Enzymes are proteins that act as the key drivers of metabolic transformations and processes in all cell-based life. Enzymes are also increasingly important ‘green’ tools for a variety of industrial processes, including the make and manufacture of pharmaceuticals, materials, fuels, and foods. Despite significant advances in computational protein design, the creation of useful, efficient, artificial enzymes remains a major challenge. This is due to the need to fashion proteins that are capable of simultaneously 1) holding a substrate (or binding) and 2) modifying the substrate (or catalysis). The proposed experiments will test a potentially generalizable strategy for enzyme design that separates substrate binding and catalysis for peptide-based substrates. This work employs cutting edge technologies and will provide rich training grounds for graduate students in high-throughput experimentation, DNA-encoded technology, computational protein design, and machine learning and artificial intelligence. These methods will also form the foundations of a focused outreach program to bring emerging concepts in biotechnology to the broader public. Ultimately, if successful, this research has the potential to open up new avenues for the design of enzyme function and enable the biocatalytic synthesis of peptides with new properties and biomaterials applications.This research project seeks to quantitatively characterize efficiency gains in designer peptide post-translational modification (PTM) enzymes comprised of separate substrate recruitment and modification domains. This constitutes a potentially generalizable approach to creating artificial PTM enzymes. Rosetta design suite will be used to fashion designer interfaces between the fyn-SH3 substrate recruitment domain and two different catalytic domains for comparison against naive (or undesigned) fusions as well as the parent, wild-type catalytic subunit in kinetic assays. Generalizability and flexibility of the strategy will be measured by an innovative, mRNA display-based substrate display assay approach and results analyzed and integrated with advanced machine learning (ML) methods. These model enzymes will further be employed to assess the additive effect of the design strategy on small, designer biosynthetic pathways.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学部生命过程化学（CLP）项目的支持下，阿尔伯特·A·埃克塞特博士（Dr. Albert A.来自查佩尔山的北卡罗来纳州大学的鲍尔斯将研究制造人工酶的方法。 酶是蛋白质，在所有以细胞为基础的生命中充当代谢转化和过程的关键驱动因素。 酶也是越来越重要的“绿色”工具，用于各种工业过程，包括药品、材料、燃料和食品的制造和生产。 尽管在计算蛋白质设计方面取得了重大进展，但创造有用，有效的人工酶仍然是一个重大挑战。 这是由于需要形成能够同时1）保持底物（或结合）和2）修饰底物（或催化）的蛋白质。 拟议的实验将测试一个潜在的可推广的策略，酶的设计，分离底物结合和催化肽为基础的基板。 这项工作采用了尖端技术，将为研究生在高通量实验，DNA编码技术，计算蛋白质设计，机器学习和人工智能方面提供丰富的培训基础。 这些方法也将形成一个重点推广计划的基础，将生物技术的新兴概念带给更广泛的公众。 最终，如果成功的话，这项研究有可能为酶功能的设计开辟新的途径，并使生物催化合成的肽与新的性能和生物材料application.This研究项目旨在定量表征设计师肽翻译后修饰（PTM）酶的效率增益组成的单独的底物招募和修饰域。 这构成了产生人工PTM酶的潜在可推广的方法。 Rosetta设计套件将用于设计fyn-SH 3底物募集结构域和两个不同催化结构域之间的界面，用于在动力学测定中与幼稚（或未设计的）融合体以及亲本野生型催化亚基进行比较。 该策略的通用性和灵活性将通过创新的基于mRNA展示的底物展示分析方法进行测量，并将结果与先进的机器学习（ML）方法进行分析和整合。 这些模型酶将进一步用于评估设计策略对小型设计师生物合成途径的加性效应。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

项目成果

Designer installation of a substrate recruitment domain to tailor enzyme specificity.

设计师安装底物招募域来定制酶特异性。

• DOI: 10.1038/s41589-022-01206-0
• 发表时间: 2023
• 期刊: Nature chemical biology
• 影响因子: 14.8
• 作者: Park,Rodney;Ongpipattanakul,Chayanid;Nair,SatishK;Bowers,AlbertA;Kuhlman,Brian
• 通讯作者: Kuhlman,Brian

Tyrosinase-Catalyzed Peptide Macrocyclization for mRNA Display

用于 mRNA 显示的酪氨酸酶催化肽大环化

• DOI: 10.1021/jacs.2c12629
• 发表时间: 2023
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Fleming, Matthew C.;Bowler, Matthew M.;Park, Rodney;Popov, Konstantin I.;Bowers, Albert A.
• 通讯作者: Bowers, Albert A.