Synthetic Protein Chemistry

描述(由申请人提供)：通过定点突变系统地修改蛋白质的能力改变了蛋白质科学，使结构功能研究能够以常规方式进行。然而，尽管这些方法实用，但对以与正常细胞核糖体表达不相容的方式修饰的蛋白质的需求日益增长。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几乎没有限制。因此，合成的蛋白质包含了非天然的氨基酸侧链、主干元件、特定位点的翻译后修饰和生物物理探针，为了解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的有机分子的挑战，这些策略促进了来自固相多肽合成的未受保护的多肽的结合。然而，尽管这些方法证明了这些方法的实用性，但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才能实践。在这项建议中，我们描述了一个全面的战略，以完善现有的合成蛋白质化学工具，并引入新的合成方法，目的是开发高效和高产率的蛋白质合成。具体地说，我们建议开发新型的含硒氨基酸和N-末端辅助基团，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的固相法合成亲电性硫代酯多肽的方法。总而言之，这些方法学的进步有望显著扩大化学在蛋白质科学中解决问题的使用。我们将测试这些方法在合成包括二氢叶酸还原酶、单链抗体和α-突触核蛋白在内的几种蛋白质中的有效性。总体而言，我们的目标是开发和改进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稳健和通用的方法，将非编码探针引入到具有生物学意义和合成挑战性的蛋白质系统中。

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.