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.[

尽管体外定点诱变在通过靶向氨基酸交换研究蛋白质结构和功能方面具有不可估量的价值，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，非天然氨基酸为通过探测氨基酸周围的空间来研究蛋白质结构和功能提供了丰富的试剂来源。通过改造大肠杆菌来接受正交氨酰基 tRNA 合成酶/tRNA 对，从而允许通过翻译抑制引入不寻常的氨基酸，这种遗传密码的扩展已经成为可能。或者，耦合转录/翻译系统被设计用于利用带化学电荷的 tRNA。与 NCI/SAIC 蛋白质表达实验室合作，高效的无细胞翻译系统与新型抑制 tRNA 技术相结合，可将非天然氨基酸类似物定点插入 p66/p51 HIV-1 RT 的 p66 亚基中（西塔拉曼等人，2003）。使用这种方法（Klarmann 等，2004），m-氟-Tyr 和正-Tyr 被替换为 DNA 聚合酶 -Tyr-Met-Asp-Asp- 活性位点基序的 Tyr183，后者导致丢失RNA 依赖性 DNA 聚合酶活性不受影响，而 DNA 依赖性 DNA 聚合酶活性不受影响。随后，p66 RT 的 Tyr115 和 Tyr183 被巯基-Tyr、1-萘酚-Tyr、2-萘酚-Tyr、间-Tyr 和甲氨基-Tyr 取代。初步数据表明，在 115 位引入甲基氨基-Tyr 后，会诱导对核苷类似物 3TC 的抗性。重组和纯化 p66 亚基含有氨基酸类似物的 HIV-1 RT 的能力将允许在分辨率水平超过了传统定点诱变所获得的分辨率水平。目前的工作重点是引入非天然氨基酸来研究 HIV-1 RT 残基与其核酸底物之间堆积相互作用的作用。