Near-natural amino acid mutagenesis for the study of protein function

用于蛋白质功能研究的近天然氨基酸诱变

• 批准号: 0848398
• 负责人: Thomas Cropp
• 金额: $ 48.6万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2011-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0848398&HistoricalAwards=false
• 关键词: Near; natural; amino; acid; mutagenesis

The Chemistry Division and the Division of Molecular and Cellular Biosciences, both at the National Science Foundation, support a collaborative research project between Professor Thomas A. Cropp of the University of Maryland and Professor Alexander Deiters of North Carolina State University. These researchers will chemically synthesize a series of photocaged near-natural amino acids, incorporate these caged amino acids into proteins using an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair, and finally, test the ability of these amino acids to precisely modulate enzymatic function, elucidating reaction mechanisms. The project hopes to address some of the fundamental problems of using near-natural amino acids as probes for protein structure and function. The approach, if successful, can provide a general tool for inserting near-natural amino acids specifically into proteins of interest. The goals of this study are to understand the H-bonding network of glutathione-S-transferase, green fluorescent protein (GFP), and the catalytic mechanism of choline oxidase.This award by the Chemistry Division and the Division of Molecular and Cellular Biosciences provides support for a unique and cross-disciplinary combination of skills between Professors Cropp and Dieters that involve protein engineering and synthetic chemistry, which will offer fantastic training opportunities for undergraduate and graduate students at both schools. The interactions between students from the two groups will be electronically assisted by the use of a dedicated Wiki-based website. To engage the general public, the Cropp and Deiters labs will invite high school teachers to conduct summer research on near-natural amino acid mutagenesis. The information gained from working with non-scientists on this research will be used to implement near-natural amino acid mutagenesis experiments in an upper-level undergraduate course. In addition, the developed research tools will benefit, and be disseminated to, enzymologists and protein chemists. Most importantly, these tools can be applied in any laboratory with standard Molecular Biology equipment and expertise, and thus will have a significant impact on the understanding of enzyme mechanisms, not possible with any other methods. From an applied perspective, some of the amino acids created in this work may expand protein function giving rise to better and more stable catalysts.

美国国家科学基金会的化学部和分子与细胞生物科学部支持马里兰大学 Thomas A. Cropp 教授和北卡罗来纳州立大学 Alexander Deiters 教授之间的合作研究项目。这些研究人员将化学合成一系列光笼近天然氨基酸，使用工程正交氨酰-tRNA合成酶/tRNA对将这些笼氨基酸整合到蛋白质中，最后测试这些氨基酸精确调节酶功能的能力，阐明反应机制。该项目希望解决使用近天然氨基酸作为蛋白质结构和功能探针的一些基本问题。该方法如果成功，可以提供一种通用工具，用于将近天然氨基酸特异性插入感兴趣的蛋白质中。这项研究的目标是了解谷胱甘肽-S-转移酶、绿色荧光蛋白（GFP）和胆碱氧化酶的催化机制的氢键网络。该奖项由化学部和分子与细胞生物科学部颁发，为 Cropp 教授和 Dieters 教授之间涉及蛋白质工程和合成化学的独特的跨学科技能组合提供了支持，这将提供出色的培训 两所学校的本科生和研究生都有机会。两个小组的学生之间的互动将通过使用专门的基于维基的网站以电子方式进行。为了吸引公众，克罗普和戴特斯实验室将邀请高中教师进行近天然氨基酸诱变的夏季研究。通过与非科学家合作进行这项研究获得的信息将用于在高年级本科课程中实施近天然的氨基酸诱变实验。此外，所开发的研究工具将使酶学家和蛋白质化学家受益并得到传播。最重要的是，这些工具可以应用于任何具有标准分子生物学设备和专业知识的实验室，因此将对酶机制的理解产生重大影响，这是任何其他方法不可能实现的。从应用的角度来看，这项工作中创建的一些氨基酸可能会扩展蛋白质功能，从而产生更好、更稳定的催化剂。