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

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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.