PROTEINS CARRYING ONE OR MORE UNNATURAL AMINO ACIDS

描述（由申请人提供）： 本提案的主要目的是开发用于将一个或多个氨基酸类似物位点特异性插入真细菌和真核生物中的蛋白质中的方法。待使用的氨基酸类似物可以包括可光活化的那些、荧光的那些、携带反应性侧链如酮基和叠氮基的那些、重原子如碘的那些、光谱探针和模拟磷酸-氨基酸的那些。除了提供生产具有新的化学和生物学性质的蛋白质的方法之外，携带此类氨基酸类似物的蛋白质将在生物学中具有广泛的应用，包括蛋白质的折叠、结构、稳定性和功能的研究，蛋白质-蛋白质相互作用，蛋白质定位和体内蛋白质动力学，晶体结构分析和信号转导。 提出了两种方法。第一种是使用琥珀抑制转移RNA（tRNA），在突变氨酰-tRNA合成酶的帮助下，在体内用氨基酸类似物氨酰化，将氨基酸类似物插入蛋白质的特定位点。第二种更普遍的方法是基于研究者实验室的发现，即抑制性tRNA，包括那些氨酰化的tRNA，可以输入哺乳动物细胞。这一发现开辟了进口抑制tRNA氨酰化在体外与各种氨基酸类似物的位点特异性插入一个或多个氨基酸类似物到蛋白质的可能性。 具体目标是：（1）分离（i）酵母酪氨酰-tRNA合成酶的突变体和（ii）E.大肠杆菌氨酰-tRNA合成酶，其用2-氨基5-酮己酸氨酰化琥珀抑制tRNA，2-氨基5-酮己酸是谷氨酰胺的类似物，其中侧链-CH 2 CH 2 CONH 2被-CH 2 CH 2 COCH 3取代。(2)鉴定、生产和纯化琥珀和赭石抑制tRNA，其不被哺乳动物氨酰-tRNA合成酶氨酰化，并且在哺乳动物细胞中具有高度抑制活性。(3)优化氨基酸类似物氨酰化的抑制性tRNA导入哺乳动物细胞的条件，并将其用于蛋白质的位点特异性插入。

DESCRIPTION (provided by applicant): The major objective of this proposal is to develop methods for the site specific insertion of one or more amino acids analogues into proteins in eubacteria and in eukaryotes. The amino acid analogues to be used could include those that are photoactivatable, those that are fluorescent, those that carry reactive side chains such as keto and azido groups, heavy atoms such as iodine, spectroscopic probes and those that mimic phospho-amino acids. Besides providing a method for the production of proteins with novel chemical and biological properties, proteins carrying such amino acid analogues will have wide applications in biology including studies on the folding, structure, stability and function of proteins, protein-protein interactions, protein localization and protein dynamics in vivo, crystallographic structure analyses and signal transduction. Two approaches are proposed. The first consists of the use of an amber suppressor transfer RNA (tRNA) aminoacylated in vivo with an amino acid analogue with the help of a mutant aminoacyl-tRNA synthetase, to insert the amino acid analogue at a specific site in a protein. A second, more general, approach is based on the finding in the investigator's laboratory that suppressor tRNAs, including those that are aminoacylated, can be imported into mammalian cells. This finding opens up the possibility of importing suppressor tRNAs aminoacylated in vitro with a variety of amino acid analogues for the site specific insertion of one or more amino acid analogues into proteins. The specific aims are: (1)To isolate mutants of (i) yeast tyrosyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with iodotyrosine, p-trifluoromethyl-diazirinylphenylalanine or p-azido phenylalanine and (ii) E. coli glutaminyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with 2-amino 5-keto hexanoic acid, an analogue of glutamine in which the side chain -CH2CH2CONH2 is replaced by - CH2CH2COCH3. (2) To identify, produce and purify amber and ochre suppressor tRNAs, which are not aminoacylated by mammalian aminoacyl-tRNA synthetases and which are highly active in suppression in mammalian cells. (3) To optimize the conditions for import of suppressor tRNAs aminoacylated with amino acid analogues into mammalian cells and to use them for site specific insertion into proteins.