Project 4

项目4，逆转录酶：RT p66/psi异源二聚体的结构研究表明，p66的结构域-结构域构型不同于psi/p5 i和p66/p66同源二聚体的结构域-结构域构型，这表明RT结构域的显著动态重排伴随异源二聚体的形成（29，30）。此外，先前的生物化学结果表明，RT中的显著构象变化对于采用与底物（例如新生DNA的s '-末端）、与dNTP和二价金属的不同相互作用模式以及对于产物释放和易位是必要的（31-35）。尽管有这样重要的运动，RT在原子水平上的溶液构象和动力学性质仍然不透明。通过使用同位素标记策略和高场/高灵敏度仪器，目前的NMR技术现在允许分析相对较大的蛋白质（NMR已确定82 kDa蛋白质折叠（36））。我们将应用溶液NMR来表征RT中的运动，使用NMR弛豫实验，由PCHPI的新成员Ishima博士开创和开发，以及基于残余偶极耦合（RDC）的方法。我们的研究将提供原子水平（或网站特定）的信息RT，信息是目前不可用的，很难获得在没有我们的混合方法。

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.