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

项目总结/摘要 在蛋白质中的所有20种天然氨基酸中，赖氨酸（Lys）经历最多样化的形式。 翻译后修饰（PTMs）。其侧链胺的独特亲核性允许Lys 用几种类型的烷基化和许多小分子和蛋白质酰化选择性修饰。 这些PTM，特别是在真核生物中，调节酶活性，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PTM的异常与 许多疾病的发展。虽然重要，但由于赖氨酸PTM的生物化学研究是繁琐的， 用它们合成蛋白质的难度。已经开发了几种方法用于合成 蛋白质与Lys PTM。但不能普遍适用。制定一个总体目标 简单的方法，通常可以应用于合成蛋白质与赖氨酸PTM，目前的 申请将集中在琥珀抑制为基础的非规范氨基酸（ncAA）的发展 诱变方法与化学转化相结合，用于将Lys PTM安装到 proteins.将追求三个具体目标：1）开发基于增强琥珀抑制的ncAA 用于重组合成具有Lys烷基化和小分子的蛋白质的诱变方法 2）开发基于琥珀抑制的ncAA诱变方法，用于合成蛋白质， 泛素和泛素样蛋白修饰;和3）结合 通过生物相容性反应来合成位点特异性地安装有两种不同的Lys PTM的蛋白质。的 成功完成拟议的研究将提供直接的方法， 蛋白质与大多数赖氨酸PTM的功能研究。

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.