SGER: Expanding the Genetic Code With Phosphoserine and Pyrrolysine

理解蛋白质结构和功能的一个基本步骤是确定多肽链中关键的单个氨基酸的精确作用。这包括与其他分子直接接触的氨基酸的作用，包括底物和配体，以及那些被修饰的氨基酸，例如通过磷酸化，作为调节蛋白质功能的手段。 在这个项目中，原核和真核系统的精确插入磷酸丝氨酸或吡咯赖氨酸在多肽链内的所需位点将被开发。 磷酸丝氨酸的插入可用于探测特定磷酸化事件对蛋白质结构和功能的影响。吡咯赖氨酸的插入可用于探测单个赖氨酸残基在配体结合和催化中的作用。 这些系统依赖于独特的古细菌tRNA合成酶/tRNA抑制子对的开发，所述古细菌tRNA合成酶/tRNA抑制子对能够在体外和体内在工程化的无义密码子处插入磷酸丝氨酸或吡咯赖氨酸。 这个项目的广泛影响是重要的，因为这个项目的成功将改变，为生物物理学，生物化学，细胞生物学，代谢生物化学和整个生命科学的其他领域的研究人员提供新的工具。 一名研究生研究员和一名高中科学教师也计划参与这项工作的各个方面。

A fundamental step in understanding protein structure and function is determining the precise roles of key, individual amino acids in the polypeptide chain. This includes the roles of amino acids involved in direct contact with other molecules, including substrates and ligands, and those that are modified, for example by phosphorylation, as a means for modulating protein function. In this project, both prokaryotic and eukaryotic systems for the precise insertion of either phosphoserine or pyrrolysine at desired sites within a polypeptide chain will be developed. The insertion of phosphoserine can be used to probe the effects of specific phosphorylation events on protein structure and function. The insertion of pyrrolysine can be used to probe the role of individual lysine residues in ligand binding and in catalysis. These systems rely on the development of unique archael tRNA synthetase/tRNA suppressor pairs capable of inserting phosphoserine or pyrrolysine at engineered, nonsense codons both in vitro and in vivo. The broader impacts of this project is significant because success in this venture would be transforming, providing new tools for researchers in biophysics, biochemistry, cell biology, metabolic biochemistry, and other fields throughout the life sciences. The participation of a graduate student researcher and a high school science teacher is planned in aspects of this work, as well.