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

国家科学基金会的化学部和分子和细胞生物科学部支持马里兰大学的托马斯·A·克罗普教授和北卡罗来纳州立大学的亚历山大·德特斯教授之间的合作研究项目。这些研究人员将通过化学方法合成一系列光笼化的近天然氨基酸，利用工程设计的正交氨酰-tRNA合成酶/tRNA对将这些笼养氨基酸结合到蛋白质中，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。如果成功，这种方法可以提供一种通用工具，将近天然的氨基酸专门插入到感兴趣的蛋白质中。这项研究的目的是为了了解谷胱甘肽-S-转移酶、绿色荧光蛋白的氢键网络以及胆碱氧化酶的催化机制。这项由化学系和分子与细胞生物科学系颁发的奖项为克罗普教授和节食者之间涉及蛋白质工程和合成化学的独特和跨学科的技能组合提供了支持，这将为两所学院的本科生和研究生提供绝佳的培训机会。来自这两个群体的学生之间的互动将通过使用专门的维基网站得到电子协助。为了让公众参与进来，Cropp和Deiters实验室将邀请高中教师在夏季进行近天然氨基酸突变的研究。在这项研究中与非科学家合作所获得的信息将被用于在一个高水平的本科课程中实施接近自然的氨基酸突变实验。此外，开发的研究工具将受益于酶学家和蛋白质化学家，并将其传播给他们。最重要的是，这些工具可以应用于任何拥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，这是任何其他方法都不可能做到的。从应用的角度来看，这项工作中创造的一些氨基酸可能会扩大蛋白质的功能，产生更好和更稳定的催化剂。

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.