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 #
5P50GM082251-07
Application #
8546397
Study Section
Special Emphasis Panel (ZRG1-AARR-K)
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
7
Fiscal Year
2013
Total Cost
$153,008
Indirect Cost
$48,732
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Zhao, Gongpu; Zhang, Peijun (2014) CryoEM analysis of capsid assembly and structural changes upon interactions with a host restriction factor, TRIM5?. Methods Mol Biol 1087:13-28
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Maertens, Goedele N; Cook, Nicola J; Wang, Weifeng et al. (2014) Structural basis for nuclear import of splicing factors by human Transportin 3. Proc Natl Acad Sci U S A 111:2728-33
Hou, Guangjin; Gupta, Rupal; Polenova, Tatyana et al. (2014) A Magic-Angle Spinning NMR Method for the Site-Specific Measurement of Proton Chemical-Shift Anisotropy in Biological and Organic Solids. Isr J Chem 54:171-183
Suiter, Christopher L; Paramasivam, Sivakumar; Hou, Guangjin et al. (2014) Sensitivity gains, linearity, and spectral reproducibility in nonuniformly sampled multidimensional MAS NMR spectra of high dynamic range. J Biomol NMR 59:57-73
Mitra, Mithun; Hercík, Kamil; Byeon, In-Ja L et al. (2014) Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties. Nucleic Acids Res 42:1095-110
Ishima, Rieko (2014) A probe to monitor performance of ¹?N longitudinal relaxation experiments for proteins in solution. J Biomol NMR 58:113-22

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