Synthetic Protein Chemistry

合成蛋白质化学

基本信息

批准号：
8163200
负责人：
PHILIP E DAWSON
金额：
$ 36.01万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8163200
关键词：
Address Amides Amino Acids Antibodies Area Biological Chemicals Chemistry Complement DHFR gene Development Dihydrofolate Reductase Elements Enzymes Excision Fluorescence Functional RNA Goals Investigation Label Laboratories Ligation Link Methodology Methods Molecular Mutagenesis N-terminal Peptide Fragments Peptide Synthesis Peptides Phase Physical condensation Post-Translational Protein Processing Protein Biosynthesis Protein Chemistry Proteins Reaction Science Selenium Series Side Site Site-Directed Mutagenesis Solid Solutions Specialist Spectrum Analysis Structure Sulfhydryl Compounds System Techniques Technology Testing Vertebral column alpha synuclein aqueous base design innovation insight interest novel peptide chemical synthesis polypeptide protein aminoacid sequence protein complex protein function protein structure protein structure function single molecule synthetic protein synuclein thioester tool

项目摘要

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. PUBLIC HEALTH RELEVANCE: Synthetic protein chemistry combines the tools of solid phase peptide synthesis and chemoselective ligation reactions to enable the total synthesis of proteins. Using these techniques, proteins can be generated that are free of the biological constraints normally place on protein structure. In this proposal we aim to develop new chemical methods for the synthesis of proteins and to apply them to challenging protein targets to interrogate the utility of the chemistry and to facilitate new investigations into the molecular basis of protein function.

描述（由申请人提供）：通过定位的诱变可以系统地修饰蛋白质的能力改变了蛋白质科学，从而使结构 - 功能研究能够以常规方式进行。然而，尽管这些方法有用，但对蛋白质的需求不断增长，这些蛋白质以与正常细胞核糖体表达不相容的方式进行了修饰。化学蛋白质合成对于这些研究具有吸引力，因为可以引入结构扰动的局限性很少。结果，蛋白质已合成，结合了不自然的氨基酸侧链，骨干元素，特定于位点的转化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。我们已经通过制定化学连接策略来解决这些大型有机分子的挑战，这些化学连接策略有助于促进源自固相肽合成的未保护肽的结合。然而，尽管这些方法具有效用，但合成蛋白化学仍然是一项艰巨的努力，而实验室数量相对较少。在该提案中，我们描述了一种完善的策略，以完善合成蛋白化学的现有工具并引入新型合成方法，以开发有效且高屈服的蛋白质合成。具体而言，我们提出了含有氨基酸和N末端辅助组的新型硒的发展，以促进水溶液中未保护多肽的化学选择性连接。为了补充这些方法，我们使用基于FMOC的固相肽合成方法进一步开发了一种用于亲电硫酯肽的方法。这些方法论上的进步共同有望大大扩展化学的使用来解决蛋白质科学中的问题。我们将测试这些方法在合成几种蛋白质（包括二氢叶酸还原酶，单链抗体和α突触核蛋白）中的效用。总体而言，我们旨在开发和完善创新的合成蛋白化学方法，目的是使蛋白质合成成为一种可靠和一般的方法，用于将非编码探针引入生物学有趣且合成具有挑战性的蛋白质系统中。公共卫生相关性：合成蛋白化学结合了固相肽合成和化学选择性连接反应的工具，以实现蛋白质的总合成。使用这些技术，可以生成蛋白质，这些蛋白质不含通常放在蛋白质结构上的生物学约束。在此提案中，我们旨在开发新的化学方法来合成蛋白质，并将其应用于具有挑战性的蛋白质靶标，以询问化学的效用，并促进对蛋白质功能分子基础的新研究。