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

摘要 从历史上看，底物和/或抑制剂与酶之间的相互作用是从结合的角度来看待的 从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 揭示了许多蛋白质，特别是dna聚合酶，在广泛的 时间尺度。这种构象灵活性对酶活性、药物作用和耐药性至关重要。 此外，当代的结构生物学方法，如X射线结晶学，只能 分解热力学稳定物种的结构，不能影响动力学中间体。我们的 该集团已开发出尖端技术--“即时”时间分辨低温电子显微镜(EM)-- 第一次促进了新的蛋白质构象的可视化，包括那些瞬间发生的 在催化过程中。具体地说，我们开发了两种冷冻-EM方法：(I)快速化学混合 酶和底物；以及(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，支持这些措施的可行性和威力 接近了。在这项应用中，我们将应用这项技术来解决生物和临床上的相关问题 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、核苷抑制剂耐药性与DNA的关系 核糖核酸酶H(RNaseH)的合成和活性。

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.