PROTEINS CARRYING ONE OR MORE UNNATURAL AMINO ACIDS

携带一种或多种非天然氨基酸的蛋白质

• 批准号: 6843141
• 负责人: UTTAM L RAJBHANDARY
• 金额: $ 28.86万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6843141
• 关键词: acylation; aminoacid; aminoacid analog; aminoacid tRNA ligase; high performance liquid chromatography; method development; mutant; nucleic acid chemical synthesis; nucleic acid purification; nucleic acid sequence; peptide chemical synthesis; polymerase chain reaction; protein purification; protein structure function; transfer RNA; transposon /insertion element

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.

描述（由申请人提供）： 该提案的主要目标是开发将一种或多种氨基酸类似物位点特异性插入真细菌和真核生物蛋白质中的方法。所使用的氨基酸类似物可包括可光活化的氨基酸类似物、发荧光的氨基酸类似物、带有酮基和叠氮基等反应性侧链的氨基酸类似物、碘等重原子、光谱探针以及模拟磷酸氨基酸的氨基酸类似物。除了提供一种生产具有新颖化学和生物学特性的蛋白质的方法外，携带此类氨基酸类似物的蛋白质还将在生物学中具有广泛的应用，包括研究蛋白质的折叠、结构、稳定性和功能、蛋白质-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。第一种方法是在突变型氨酰基-tRNA 合成酶的帮助下，利用氨基酸类似物在体内氨酰化琥珀抑制转移 RNA (tRNA)，将氨基酸类似物插入蛋白质的特定位点。第二种更通用的方法是基于研究人员实验室的发现，即抑制性 tRNA（包括氨酰化的 tRNA）可以导入哺乳动物细胞。这一发现开启了导入在体外用多种氨基酸类似物氨酰化的抑制性tRNA的可能性，用于将一个或多个氨基酸类似物位点特异性插入蛋白质中。 具体目标是：（1）分离（i）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-二氮杂环苯丙氨酸或对叠氮基苯丙氨酸氨酰化琥珀抑制tRNA和（ii）大肠杆菌谷氨酰胺酰-tRNA合成酶，用 2-氨基 5-酮己酸氨酰化琥珀抑制 tRNA，谷氨酰胺类似物，其中侧链 -CH2CH2CONH2 被 -CH2CH2COCH3 取代。 (2) 鉴定、生产和纯化琥珀和赭石抑制tRNA，它们不被哺乳动物氨酰-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) 优化将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，并将其用于位点特异性插入蛋白质中。