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

摘要 历史上，底物和/或抑制剂与酶之间的相互作用被视为结合的角度 小分子到相对刚性的蛋白质靶标。然而，计算和实验研究已经 揭示许多蛋白质，特别是 DNA 聚合酶，在广泛的范围内进行分子运动 时间尺度。这种构象灵活性对于酶活性、药物作用和耐药性至关重要。 此外，当代结构生物学方法，例如 X 射线晶体学，只能 解析热力学稳定物质的结构，并且不能提供动力学中间体的信息。我们的 小组开发了尖端技术——“即时”时间分辨冷冻电子显微镜（EM）—— 首次促进新型蛋白质构象的可视化，包括那些瞬时发生的构象 催化过程中。具体来说，我们开发了两种 TR 冷冻电镜方法：(i) 快速化学混合 酶和底物； (ii) 利用笼状底物和/或氨基酸的光活化 残留物。我们已经开发了大量的初步数据来支持这些的可行性和力量 接近。在此应用中，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、核苷抑制剂耐药性以及DNA之间的关系 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.