Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent
Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent
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DOI：
10.1073/pnas.252550199
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发表时间：
2002-12-10
影响因子：
11.1
通讯作者：
Le Grice, SFJ
中科院分区：
文献类型：
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作者：
Kvaratskhelia, M;Miller, JT;Le Grice, SFJ
我们设计了一种高分辨率的蛋白质足迹法来剖析HIV-1逆转录酶（RT）与病毒RNA：tRNA复合物的接触。实验策略包括通过伯胺选择性试剂NHS-生物素修饰RT和RT-病毒RNA：tRNA复合物中表面暴露的赖氨酸，SDS/PAGE分离p66和p51多肽，凝胶蛋白水解，以及肽片段的比较质谱分析。这种方法可以很容易地揭示在游离RT中修饰但在核蛋白复合物中不被生物素化的赖氨酸。检查RT-DNA：DNA复合物的对照实验的结果与相同复合物的晶体结构数据非常一致。探测RT-病毒RNA：tRNA复合物显示，大多数蛋白质接触与RT-DNA：DNA和RT-RNA：DNA种类一样位于引物-模板结合裂缝中。然而，我们的足迹数据表明，p66指子结构域使额外的接触病毒RNA：tRNA特异性的这种复合物，而不是检测到的DNA：DNA。本文所述的蛋白质足迹法具有用于多蛋白质-核酸接触的高分辨率溶液结构研究的一般应用。
We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDS/PAGE separation of p66 and p51 polypepticles, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.