Project 4

项目4，逆转录酶：对RT p66/psi异源二聚体的结构研究表明，p66的结构域-结构域构型与psi/p5i和p66/p66同源二聚体不同，这表明RT结构域的显著动态重排伴随着异源二聚体的形成（29,30）。此外，之前的生化结果表明，RT的显著构象变化是必要的，以采用与底物不同的相互作用模式，如新生DNA的s'端，与dNTPs和二价金属，以及产物释放和易位（31-35）。尽管有这些重要的基本运动，但RT在原子水平上的溶液构象和动力学性质仍然不透明。目前的核磁共振技术现在可以通过使用同位素标记策略和高场/高灵敏度仪器来分析相对较大的蛋白质（核磁共振已经确定了82 kda的蛋白质折叠（36））。我们将应用溶液核磁共振来表征RT中的运动，使用核磁共振弛豫实验，由PCHPI的新成员石岛博士开创和开发，以及基于残余偶极耦合（RDC）的方法。我们的研究将提供RT的原子水平（或特定位点）信息，这些信息目前还无法获得，而且在没有我们的混合方法的情况下很难获得。

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.