"On the Fly" Time Resolved Cryo-EM Studies of Intermediate HIV-1 RT Transition States

总结 从历史上看，底物和/或抑制剂与酶之间的相互作用一直被认为是结合 小分子到相对刚性的蛋白质靶点。然而，计算和实验研究 揭示了许多蛋白质，特别是DNA聚合酶，在很宽的范围内进行分子运动。 时间尺度。这种构象柔性对于酶活性、药物作用和耐药性是至关重要的。 此外，当代的结构生物学方法，如X射线晶体学，只能 解析化学稳定物质的结构，并且不能提供动力学中间体的信息。我们 该小组开发了尖端技术-“即时”时间分辨冷冻电子显微镜（EM）-， 这是第一次，促进了新的蛋白质构象的可视化，包括那些瞬时发生的构象 在催化过程中。具体而言，我们已经开发了两种TR冷冻EM方法：（i）快速化学混合 酶和底物;和（ii）利用笼状底物和/或氨基酸的光活化 残基我们已经开发了大量的初步数据，支持这些方法的可行性和力量。 接近。在本申请中，我们将应用该技术来解决生物学和临床相关问题， HIV-1逆转录酶（RT）生物学方面的知识差距，特别关注纳入 核苷和核苷抑制剂，核苷抑制剂耐药，与DNA 合成和核糖核酸酶H（RNase H）活性。

SUMMARY Historically, interactions between substrates and/or inhibitors and enzymes have been viewed in terms of binding of small molecules to relatively rigid protein targets. However, computational and experimental studies have revealed that many proteins, in particular DNA polymerases, undergo molecular motions over a wide range of timescales. Such conformational flexibility is critical for enzymatic activity, drug action and drug resistance. Moreover, contemporary structural biology approaches, such as X-ray crystallography, only have the ability to resolve the structures of thermodynamically stable species, and cannot inform on kinetic intermediates. Our group has developed cutting-edge technology - “on-the-fly” time-resolved cryo-electron microscopy (EM) - that, for the first time, facilitates visualization of novel protein conformations, including those transiently occurring during catalysis. Specifically, we have developed two TR cryo-EM approaches: (i) rapid chemical mixing of enzyme and substrates; and (ii) photo-activation that takes advantage of caged substrates and/or amino acid residues. We have developed substantial preliminary data that support the feasibility and power of these approaches. In this application, we will apply this technology to address biologically and clinically relevant knowledge gaps in HIV-1 reverse transcriptase (RT) biology, with specific focus on the incorporation of nucleosides and nucleoside inhibitors, nucleoside inhibitor resistance, and the relationship between DNA synthesis and ribonuclease H (RNase H) activity.