Unnatural Amino Acids as Probes of RT Structure and Func

非天然氨基酸作为 RT 结构和功能的探针

• 批准号: 7291840
• 负责人: Stuart F. J. Le Grice
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7291840
• 关键词: Unnatural; Amino; Acids; Probes; RT

Although in vitro site-directed mutagenesis has been invaluable in studying protein structure and function via targeted amino acid exchanges, this approach is limited inasmuch as only the other 19 amino acids defined by the genetic code can be introduced. In contrast, unnatural amino acids provide a rich source of agents to study protein structure and function by probing the space around an amino acid. Such expansion of the genetic code has been possible through engineering E. coli to accept an orthogonal aminoacyl tRNA synthetase/tRNA pair, allowing the unusual amino acid to be introduced via translational suppression. Alternatively, coupled transcription/translation systems have been designed to exploit chemically charged tRNAs. In collaboration with the NCI/SAIC Protein Expression Laboratory, a highly efficient cell-free translation system has been coupled with a novel suppressor tRNA technology to site-specifically insert unnatural amino acid analogs into the p66 subunit of p66/p51 HIV-1 RT (Sitaraman et al., 2003). Using this approach (Klarmann et al., 2004), m-fluoro-Tyr and nor-Tyr were substituted for Tyr183 of the DNA polymerase -Tyr-Met-Asp-Asp- active site motif, the latter of which resulted in loss of RNA-dependent DNA polymerase while DNA-dependent DNA polymerase activity was unaffected. Subsequent to this, mercapto-Tyr, 1-naphthol-Tyr, 2-naphthol-Tyr, m-Tyr and methylamino-Tyr have been substituted for Tyr115 and Tyr183 of p66 RT. Preliminary data indicate that resistance to the nucleoside analog 3TC is induced following introduction of methylamino-Tyr at position 115. The ability to reconstitute and purify HIV-1 RT whose p66 subunit contains amino acid analogs will allow structure/function analysis to be performed at a level of resolution exceeding that obtained by conventional site-directed mutagenesis. Current efforts focus on the introduction of unnatural amino acids to study the role of stacking interactions between residues of HIV-1 RT and its nucleic acid substrate.[

尽管体外位置定向的诱变在研究蛋白质结构和通过靶向氨基酸交换的功能方面具有无价之宝，但这种方法受到限制，因为只能引入遗传密码定义的其他19个氨基酸。相比之下，非天然氨基酸通过探测氨基酸周围的空间来研究蛋白质结构和功能的丰富来源。通过工程大肠杆菌，可以接受正交氨基酰基tRNA合成酶/tRNA对的遗传代码的这种扩展，从而可以通过翻译抑制引入异常的氨基酸。或者，耦合的转录/翻译系统已被设计为利用化学电荷的TRNA。与NCI/SAIC蛋白表达实验室合作，一种高效的无细胞翻译系统与一种新型的抑制器TRNA技术耦合，以特异性地将非天然氨基酸类似物插入p66/p51 HIV-1 RT的P66亚基中（Sitaraman等人，2003年）。使用这种方法（Klarmann等，2004），M-fluoro-tyr和Nor-tyr被取代为DNA聚合酶-Tyr-Met-Asp-Asp-Apt-Active位点的Tyr183，后者的后者导致RNA依赖性DNA聚合酶的丧失，而DNA依赖性DNA依赖性DNA依赖性DNA依赖性DNA依赖性依赖性。此后，已将Mercapto-Tyr，1-萘酚-TYR，2-萘酚-TYR，M-TYR和甲基氨基-TYR代替为P66 RT的Tyr115和Tyr183。初步数据表明，在第115位引入甲基氨基-TYR后，诱导对核苷类似物3TC的抗性。重新构建和净化HIV-1 RT的能力，其p66亚基包含氨基酸类似物的抗性HIV-1 RT将允许以常规位置的分辨率进行结构/功能分析，以超过常规的模型发电量进行结构/功能分析。当前的努力集中在引入非自然氨基酸的引入，以研究HIV-1 RT残基与其核酸底物之间堆叠相互作用的作用。