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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Specialized Center (P50)
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Special Emphasis Panel (ZRG1-AARR-K)
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University of Pittsburgh
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