Develop General Methods for the Synthesis of Proteins with Posttranslational Lysine Modifications

开发具有翻译后赖氨酸修饰的蛋白质合成的通用方法

• 批准号: 10307614
• 负责人: Wenshe Ray Liu
• 金额: $ 33.4万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307614
• 关键词: Acylation; Affect; Alkylation; Amber; Amines; Amino Acids; Amino Acyl-tRNA Synthetases; Bacteriophages; Biochemical; Catalysis; Cells; Chemicals; Complex; Cysteine; DNA biosynthesis; Development; Disease; Engineering; Escherichia coli; Eukaryota; Evolution; Gene Expression; Genetic; Genus Mycobacterium; Health; Health Promotion; Human; Investigation; Life; Ligation; Lysine; Methods; Mission; Modification; Mutagenesis; Organism; Post-Translational Protein Processing; Protein Biosynthesis; Proteins; Public Health; Reaction; Recombinants; Regulation; Research; Side; Signal Transduction; Site; Technology; Testing; Transfer RNA; Ubiquitin; Ubiquitin Like Proteins; Ubiquitination; United States National Institutes of Health; amidation; base; biomaterial compatibility; cellular development; deep sequencing; enzyme activity; improved; protein metabolism; proteostasis; small molecule

PROJECT SUMMARY/ABSTRACT Among all 20 native amino acids in proteins, lysine (Lys) undergoes the most diverse forms of posttranslational modifications (PTMs). The unique nucleophilicity of its side chain amine allows Lys to be selectively modified with several types of alkylation and a number of small-molecule and protein acylations. These PTMs, especially in eukaryotes, regulate enzyme activities, interactions of proteins with their partners, cellular localization of proteins, and protein metabolism. Abnormality of these PTMs correlates with the development of many diseases. Although important, biochemical studies of Lys PTMs are cumbersome due to the difficulty to synthesize proteins with them. Several methods have been developed for the synthesis of proteins with Lys PTMs. However, they cannot be generally applied. With an overall objective to formulate straightforward methods that can be generally applied for the synthesis of proteins with Lys PTMs, the current application will focus on the development of amber suppression-based noncanonical amino acid (ncAA) mutagenesis methods in combination with chemical transformation for the installation of Lys PTMs into proteins. Three specific aims will be pursued: 1) Develop enhanced amber suppression-based ncAA mutagenesis methods for the recombinant synthesis of proteins with Lys alkylations and small-molecule acylations; 2) Develop amber suppression-based ncAA mutagenesis methods for the synthesis of proteins with ubiquitin and ubiquitin-like protein modifications; and 3) Formulate recombinant strategies in conjunction with biocompatible reactions to synthesize proteins installed site-specifically with two different Lys PTMs. The successful completion of the proposed study will make available straightforward methods for the synthesis of proteins with most Lys PTMs for their functional investigation.

项目摘要/摘要 在蛋白质中的所有20种天然氨基酸中，赖氨酸(Lys)经历了最多样化的形式 翻译后修饰(PTM)。其侧链胺的独特亲核性使赖氨酸能够 选择性地用几种类型的烷基化和一些小分子和蛋白质酰化来修饰。 这些PTM，特别是在真核生物中，调节酶的活性，蛋白质与其伴侣的相互作用， 蛋白质的细胞定位和蛋白质代谢。这些PTMS的异常与 许多疾病的发展。尽管Lys PTMS的生化研究很重要，但由于以下原因，生化研究很麻烦 用它们合成蛋白质的难度。已经开发了几种合成方法来合成 含有Lys PTMS的蛋白质。然而，它们不能普遍适用。有一个总体目标来制定 可以普遍应用于利用Lys PTMS合成蛋白质的简单方法，目前 应用重点是基于琥珀抑制的非典型氨基酸(NCAA)的开发 利用诱变和化学转化相结合的方法将Lys PTMS安装到 蛋白质。将追求三个具体目标：1)发展基于增强琥珀抑制的NCAA 含Lys烷基化和小分子蛋白质重组合成的诱变方法 2)开发基于琥珀抑制的NCAA突变方法，用于合成具有以下特征的蛋白质 泛素和泛素样蛋白修饰；以及3)结合 与两种不同的赖氨酸PTM进行生物相容反应，以合成特定位置安装的蛋白质。这个 拟议研究的成功完成将使合成的方法变得简单 与大多数Lys PTM的蛋白质进行功能研究。

项目成果

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli.

使用大肠杆菌中的遗传密码扩展对组蛋白进行位点特异性赖氨酸乙酰化以进行核小体重建。

• DOI: 10.3791/62113
• 发表时间: 2020
• 期刊: Journal of visualized experiments : JoVE
• 作者: Rowlett,ChesleyMarie;Liu,WensheRay
• 通讯作者: Liu,WensheRay

A Designed, Highly Efficient Pyrrolysyl-tRNA Synthetase Mutant Binds o-Chlorophenylalanine Using Two Halogen Bonds.

• DOI: 10.1016/j.jmb.2022.167534
• 发表时间: 2022-04-30
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Vatansever, Erol C.;Yang, Kai S.;Geng, Zhi Zachary;Qiao, Yuchen;Li, Pingwei;Xu, Shiqing;Liu, Wenshe Ray
• 通讯作者: Liu, Wenshe Ray

Taf2 mediates DNA binding of Taf14.

• DOI: 10.1038/s41467-022-30937-w
• 发表时间: 2022-06-08
• 期刊: Nature communications
• 影响因子: 16.6

An optimal "Click" formulation strategy for antibody-drug conjugate synthesis.

• DOI: 10.1016/j.bmc.2020.115808
• 发表时间: 2020-12-15
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: Vatansever EC;Kang J;Tuley A;Ward ES;Liu WR
• 通讯作者: Liu WR

The Pyrrolysyl-tRNA Synthetase Activity can be Improved by a P188 Mutation that Stabilizes the Full-Length Enzyme.

• DOI: 10.1016/j.jmb.2022.167453
• 发表时间: 2022-04-30
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Cho, Chia-Chuan;Blankenship, Lauren R.;Ma, Xinyu;Xu, Shiqing;Liu, Wenshe
• 通讯作者: Liu, Wenshe