Abstract

The goal of this proposal is to investigate protein-ligand and ? protein-protein interactions in HIV-1 reverse transcriptase (RT). Being a key player in HIV infection, RT is the molecular target of the majority of AIDS drugs. Ligands studied will include five nonnucleoside reverse transcriptase inhbitiors (NNRTI) as well as normal substrates. RTs studied will include up to five drug resistance mutants in addition to wild type. The enzyme copies the single-stranded genomic RNA into a double-stranded DMA provirus, which is subsequently integrated into the host chromosome by HIV integrase. RT has RNA- and DMA-dependent DMA polymerase and RNase H activities. The biologically active enzyme is a heterodimer of p66 and p51 subunits. The p51 subunit is derived from p66 subunit by HIV protease. The proposal applies biochemical and biophysical techniques, including powerful site-specific fluorescence techniques, to fundamental investigations of proposed inhibition mechanisms and protein conformations that may reveal new targets for antiviral therapy. This laboratory is presently one of few using solution biophysical techniques to study RT. Specifically, we propose to: 1. Characterize inhibitor binding to RT. 2. Determine inhibitor effects on individual steps in DMA polymerization. 3. Investigate inhibitor effects on conformation and dynamics of RT subunits and assembly. ? ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071267-02
Application #
7197365
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Basavappa, Ravi
Project Start
2006-03-15
Project End
2010-02-28
Budget Start
2007-03-01
Budget End
2008-02-29
Support Year
2
Fiscal Year
2007
Total Cost
$237,785
Indirect Cost

  Institution

Name
Case Western Reserve University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106

  Publications

Seckler, James M; Barkley, Mary D; Wintrode, Patrick L (2011) Allosteric suppression of HIV-1 reverse transcriptase structural dynamics upon inhibitor binding. Biophys J 100:144-53
Braz, Valerie A; Holladay, Leslie A; Barkley, Mary D (2010) Efavirenz binding to HIV-1 reverse transcriptase monomers and dimers. Biochemistry 49:601-10
Braz, Valerie A; Barkley, Mary D; Jockusch, Rebecca A et al. (2010) Efavirenz binding site in HIV-1 reverse transcriptase monomers. Biochemistry 49:10565-73
Seckler, James M; Howard, Kathryn J; Barkley, Mary D et al. (2009) Solution structural dynamics of HIV-1 reverse transcriptase heterodimer. Biochemistry 48:7646-55
Venezia, Carl F; Meany, Brendan J; Braz, Valerie A et al. (2009) Kinetics of association and dissociation of HIV-1 reverse transcriptase subunits. Biochemistry 48:9084-93
Braz, Valerie A; Howard, Kathryn J (2009) Separation of protein oligomers by blue native gel electrophoresis. Anal Biochem 388:170-2