Unnatural Amino Acids as Probes of RT Structure and Func

虽然体外定点突变在通过靶向氨基酸交换研究蛋白质结构和功能方面有很大的价值，但这种方法是有限的，因为只有遗传密码定义的其他19个氨基酸可以被引入。相比之下，非天然氨基酸提供了丰富的物质来源，通过探测氨基酸周围的空间来研究蛋白质的结构和功能。这种遗传密码的扩展是可能的，通过改造大肠杆菌接受正交的氨基酰基tRNA合成酶/tRNA对，允许通过翻译抑制引入这种不寻常的氨基酸。或者，已经设计了耦合的转录/翻译系统来利用带化学电荷的tRNA。与NCI/SAIC蛋白质表达实验室合作，一个高效的无细胞翻译系统与一种新的抑制tRNA技术相结合，将非自然氨基酸类似物定点插入到p66/p51 HIV-1 RT的p66亚单位中(Sitaraman等人，2003年)。使用这种方法(Klarmann等人，2004年)，m-氟-Tyr和Nor-Tyr取代了DNA聚合酶-Tyr-Met-Asp-Asp活性位点基序的Tyr183，后者导致RNA依赖的DNA聚合酶丢失，而DNA依赖的DNA聚合酶活性不受影响。此后，p66 RT中的Tyr115和Tyr183分别被硫醇酪氨酸、1-萘酚-酪氨酸、2-萘酚-酪氨酸、m-酪氨酸和甲氨基-酪氨酸取代。初步数据表明，在第115位引入甲氨基-酪氨酸后，诱导了对核苷类似物3TC的抗性。重组和纯化p66亚基含有氨基酸类似物的HIV-1RT的能力将使结构/功能分析的分辨率超过传统的定点突变。目前的工作主要集中在引入非天然氨基酸来研究HIV-1RT残基与其核酸底物之间的堆积作用。

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.[