Probing the Active Site Flexibility of HIV-RT

Omid Khakshoor, Stanford University, Palo Alto
Mentor: Eric Kool
Training in Basic Biomedical Sciences
Postdoctoral Fellowship Award

This study aims to determine if the thymidine 5-methyl group influences the efficiency and fidelity of DNA replication through Van der Waals interactions with amino acid side chains in the HIV-1 Reverse Transcriptase active site. A series of variably sized 5-substituted thymidine analogs will provide useful probes to study the effects of the 5-substituent’s size and the corresponding Van der Waals interactions on DNA replication. These probes will also determine if the lack of the 5-methyl group in uridine is responsible for the lower fidelity of RNA-dependent DNA replication by HIV-1 reverse transcriptase (HIV-1 RT), as compared to DNA-dependent DNA replication by the same enzyme. This research is important because HIV-1 RT is vital in replication of the AIDS viral genome, and the low fidelity in RNA-dependent DNA replication by this enzyme is a major contributor to the rapid growth of drug-resistant mutants of HIV. Most current treatments for HIV infections are based on compounds that partially inhibit the function of HIV-1 RT. Recognizing the lack of the 5-methyl group in uridine as the chief reason of the lower fidelity associated with the RNA-dependent activity can help researchers to design more efficient inhibitors for this enzyme as more powerful therapeutics against AIDS.

This research will also be beneficial to my career training at the chemistry and biology interface. During my graduate studies, my research aimed to better understand protein-protein interactions through mimicry of β-sheet component of protein structures. This postdoctoral training will allow me to study DNA-protein interactions through mimicry of nucleotide components of DNA and RNA structures. During this study, I will become familiar with the chemical and physical behaviors of DNA and RNA. I will learn how to synthesize DNA and RNA nucleotide derivatives and oligonucleotides. I will learn to run enzymatic assays and determine the kinetic parameters corresponding to enzymatic activity, and I will be trained to calculate thermodynamic parameters associated with the stability of DNA and RNA helices.