Synthetic Protein Chemistry

描述（由申请人提供）：通过位点定向诱变系统地修饰蛋白质的能力已经改变了蛋白质科学，使结构-功能研究能够以常规方式进行。然而，尽管这些方法具有实用性，但对以与正常细胞核糖体表达不相容的方式进行修饰的蛋白质的需求越来越大。化学蛋白质合成对这些研究很有吸引力，因为可以引入的结构扰动很少有限制。因此，蛋白质的合成结合了非天然氨基酸侧链、主干元件、位点特异性翻译后修饰和生物物理探针，并为蛋白质功能的分子基础提供了许多独特的见解。我们已经解决了通过开发化学连接策略来合成这些大型有机分子的挑战，这些策略促进了来自固相肽合成的无保护肽的结合。然而，尽管这些方法的实用性得到了证明，合成蛋白质化学仍然是一项具有挑战性的努力，只有相对少数的实验室进行了实践。在本提案中，我们描述了一种综合策略，以改进现有的合成蛋白质化学工具，并引入新的合成方法，目的是开发高效高产的蛋白质合成。具体来说，我们建议开发新的含硒氨基酸和n端辅助基团，以促进水溶液中无保护多肽的化学选择性连接。为了补充这些方法，我们进一步开发了一种基于Fmoc的固相肽合成方法来合成亲电性硫酯肽。总之，这些方法上的进步有望极大地扩展化学在解决蛋白质科学问题方面的应用。我们将测试这些方法在几种蛋白质合成中的实用性，包括二氢叶酸还原酶、单链抗体和α -突触核蛋白。总的来说，我们的目标是开发和完善创新的合成蛋白质化学方法，目标是使蛋白质合成成为一种强大而通用的方法，用于将非编码探针引入生物学上有趣的和具有合成挑战性的蛋白质系统。

DESCRIPTION (provided by applicant): The ability to systematically modify proteins through site directed mutagenesis has transformed protein science, enabling structure-function studies to be performed in a routine manner. However, despite the utility of these methods, there is a growing demand for proteins that are modified in a manner incompatible with normal cellular ribosomal expression. Chemical protein synthesis is attractive for these studies since there are very few limitations on the structural perturbations that can be introduced. As a result, proteins have been synthesized incorporating unnatural amino acid side chains, backbone elements, site-specific post- translational modifications and biophysical probes and have provided numerous unique insights into the molecular basis of protein function. We have addressed the challenge of synthesizing these large organic molecules through the development of chemical ligation strategies that facilitate the conjugation of unprotected peptides derived from solid phase peptide synthesis. However, despite the demonstrated utility of these methods, synthetic protein chemistry remains a challenging endeavor that is practiced by a relatively few number of laboratories. In this proposal, we describe a comprehensive strategy to refine the existing tools of synthetic protein chemistry and introduce novel synthetic approaches with the aim of developing efficient and high yielding syntheses of proteins. Specifically, we propose the development of novel selenium containing amino acids and N-terminal auxiliary groups to facilitate the chemoselective linking of unprotected polypeptides in aqueous solution. To complement these methods, we further develop an approach for the electrophilic thioester peptides using Fmoc based solid phase peptide synthesis approaches. Together, these methodological advances promise to significantly extend the use of chemistry to address questions in protein science. We will test the utility of these methods in the synthesis of several proteins including dihydrofolate reductase, single chain antibodies and alpha synuclein. Overall, we aim to develop and refine innovative synthetic protein chemistry methods with the goal of making protein synthesis a robust and general methodology for the introduction of non-coded probes into biologically interesting and synthetically challenging protein systems.