PROTEINS CARRYING ONE OR MORE UNNATURAL AMINO ACIDS

描述(由申请人提供)： 这项建议的主要目标是开发一种或多种氨基酸类似物在真细菌和真核生物中的蛋白质中定位插入的方法。要使用的氨基酸类似物可以包括那些可光激活的，那些是荧光的，那些带有反应性侧链的，如酮和叠氮基，重原子，如碘，光谱探针，以及那些模拟磷酸-氨基酸的。除了提供一种制备具有新的化学和生物学性质的蛋白质的方法外，携带这种氨基酸类似物的蛋白质还将在生物学中有广泛的应用，包括研究蛋白质的折叠、结构、稳定性和功能、蛋白质与蛋白质的相互作用、蛋白质在体内的定位和蛋白质动力学、晶体结构分析和信号转导。 提出了两种方法。第一种方法是使用琥珀抑制物转移RNA(TRNA)，在体内与氨基酸类似物在突变的氨基酰-tRNA合成酶的帮助下，将氨基酸类似物插入蛋白质的特定位置。第二种更一般的方法是基于研究人员实验室的发现，即抑制tRNA，包括那些氨基酰化的tRNA，可以输入哺乳动物细胞。这一发现为在体外与各种氨基酸类似物进行氨酰化的抑制tRNAs的导入提供了可能性，以便将一个或多个氨基酸类似物特异性地插入蛋白质中。 具体目标是：(1)分离酵母酪氨酰-tRNA合成酶的突变体，该酶用碘酪氨酸、对三氟甲基-重氮基苯丙氨酸或对叠氮苯丙氨酸来氨基酰化琥珀酸抑制物tRNA，以及(Ii)用2-氨基5-酮基己酸氨基酰化琥珀酰化琥珀酸抑制物tRNA的大肠杆菌谷氨酰基-tRNA合成酶。(2)鉴定、制备和纯化在哺乳动物细胞中具有高度抑制活性的琥珀和赭石抑制因子tRNAs，这些tRNAs在哺乳动物细胞中不被氨基酰-tRNA合成酶氨酰化。(3)优化用氨基酸类似物修饰的抑制子tRNAs导入哺乳动物细胞的条件，并将其用于位点特异性插入蛋白质。

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.