Project 4

项目4

• 批准号: 8528173
• 负责人: RIEKO ISHIMA
• 金额: $ 45.7万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8528173
• 关键词: Adopted; Affinity; Amino Acids; Binding; Biochemical; Characteristics; Chemicals; Coupling; DBL Oncoprotein; DNA; DNA Sequence Rearrangement; Data; HIV; Hybrids; Isotope Labeling; Label; Ligand Binding; Molecular Conformation; Motion; Physiologic pulse; Property; Proteins; RNA-Directed DNA Polymerase; Relaxation; Residual state; Ribonuclease H; Roentgen Rays; Sampling; Shapes; Signal Transduction; Site; Solutions; Structure; System; Technology; Temperature; Thumb structure; Vertebral column; Virion; base; divalent metal; inhibitor/antagonist; insight; instrument; meetings; member; methionine methyl ester; monomer; mutant; nanosecond; novel; protein folding; research study; solid solution; solid state; solid state nuclear magnetic resonance

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.

项目4，逆转录酶：RT p66/PSI异二聚体的结构研究表明，p66的域 - 域构型与PSI/p5i和p66/p66同型二聚体的结构构型不同，这表明RT域的显着动态重排伴有RT域伴随杂志杂质形成（29,30）。此外，先前的生化结果表明，RT的显着构象变化对于采用与底物的不同相互作用模式是必要的，例如新生DNA的S'-末端，带有DNTPS和DNTPS和Divalent Metals，以及产品释放和易位（31-35）。尽管基本上重要的动作，但原子水平的RT的溶液构象和动态特性仍然不透明。当前的NMR技术现在可以通过使用同位素标记的策略和高视野/高敏化仪器来确定相对较大的蛋白质（由NMR（36））确定的相对较大蛋白（82 kDa蛋白折叠）。我们将使用NMR弛豫实验（由PCHPI的新成员Ishima博士以及残留的偶性偶联（RDC）基于基于的方法进行开发和开发，我们将使用溶液NMR来表征RT中的运动。我们的研究将提供有关RT的原子水平（或特定地点）信息，这些信息目前尚不可用，并且在没有混合方法的情况下很难获得。