In vivo Incorporation of Unnatural Amino Acids

描述(申请人提供)：在之前的资助期间，我们开发了一种通用方法，允许人们系统地将具有新的物理、化学和生物特性的氨基酸添加到大肠杆菌和酵母的遗传密码中。这些氨基酸是由琥珀密码子标签和四重密码子Agga高效和高保真地遗传编码的。在这个提议中，我们将从几个方面扩展这项工作：(1)我们将解决几种编码非天然氨基酸的E.线圈和酵母正交合成酶的三维晶体结构，以更好地了解如何进化合成酶的选择性；(2)我们将在E.线圈和酵母中遗传编码一些N-和O-连接的单糖，并证明它们可以用来产生选择性糖基化的蛋白质；(3)我们将开发一种健壮和通用的方法来产生tRNA-合成酶对，可以在哺乳动物细胞中插入非天然氨基酸；(4)我们将在大肠杆菌和酵母中对一些生物物理探针进行基因编码(包括荧光氨基酸、自旋标记氨基酸、光笼氨基酸、含氨基酸的重原子，以及可用作选择性核磁共振和红外探针的氨基酸)。这些研究将极大地扩展我们操纵蛋白质结构的能力，以创造具有新功能的蛋白质，并在体外和体内探索蛋白质的结构和功能。

DESCRIPTION (provided by applicant): In the previous funding period we developed a general method that allows one to systematically add amino acids with novel physical, chemical and biological properties to the genetic code of E. coil and yeast. These amino acids are genetically encoded efficiently and with high fidelity by the amber codon TAG and quadruplet codon AGGA. In this proposal we will extend this work in several directions: (1) We will solve the three dimensional crystal structures of several E. coil and yeast orthogonal synthetases that encode unnatural amino acids to better understand how to evolve synthetase selectivity; (2) We will genetically encode a number of N- and O-linked monosaccharides in E. coil and yeast and demonstrate that they can be used to generate selectively glycosylated proteins; (3) We will develop a robust and general method for generating tRNA - synthetase pairs that can insert unnatural amino acids in mammalian cells; (4) We will genetically encode a number of biophysical probes in E. coil and yeast (including fluorescent amino acids, spin-labeled amino acids, photocaged amino acids, heavy atoms containing amino acids, and amino acids that can be used as selective NMR and IR probes). These studies should dramatically expand our ability to manipulate protein structure in order to create proteins with new functions and to probe protein structure and function in vitro and in vivo.