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

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。

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.