Project 4, Reverse Transcriptase: Structural studies of the RT p66/psi heterodimer indicate that the domain-domain configuration of p66 is different from that of the psi/p5i and p66/p66 homodimers, suggesting that significant dynamical rearrangements of the RT domains accompany heterodimer formation(29,30). Furthermore, previous biochemical results imply that significant conformational changes in RT are necessary to adopt distinct interaction modes with substrate, such as the s'-terminus of nascent DNA, with dNTPs and divalent metals, and for product-release, and translocation(31-35). Despite such fundamentally important motions, the solution conformation and dynamic properties of RT at the atomic level are still opaque. Current NMR technology now permits analyses of relatively large proteins (an 82-kDa protein fold has been determined by NMR(36)), through the use of isotope-labeling strategies and high-field/high-sensitivity instruments. We will apply solution NMR to characterize motions in RT, using NMR relaxation experiments, pioneered and developed by Dr. Ishima, a new member of the PCHPI, as well as residual dipolar coupling (RDC)-based approaches. Our studies will provide atomic level (or site specific) information on RT, information that is not currently available and difficult to obtain in the absence of our hybrid approach.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center (P50)
Project #
5P50GM082251-08
Application #
8727032
Study Section
Special Emphasis Panel (ZRG1-AARR-K)
Project Start
Project End
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
8
Fiscal Year
2014
Total Cost
$158,556
Indirect Cost
$50,499
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik et al. (2016) The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes. J Biol Chem 291:11809-19
Ramalho, Ruben; Rankovic, Sanela; Zhou, Jing et al. (2016) Analysis of the mechanical properties of wild type and hyperstable mutants of the HIV-1 capsid. Retrovirology 13:17
Oum, Yoon Hyeun; Desai, Tanay M; Marin, Mariana et al. (2016) Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion. J Virol Methods 233:62-71

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