KEY INTERACTIONS FOR HIV 1 RT STABILITY AND DIMERIZATION

描述：本提案的主要重点是鉴定氨基 酸的相互作用是形成稳定的HIV-1逆转录病毒的关键 转录酶（RT）异二聚体。对这些相互作用的详细了解 将潜在地为合理设计新类别的 治疗上有用的抑制剂，用于涉及形成 活性HIV-1 RT。拟议的实验将区分相互作用 对于1）蛋白质折叠，2）折叠蛋白质结构的稳定性， 和3）p66/p51异二聚体的形成。识别方法 肽抑制剂的合成和成熟的各个阶段， 还将研究RT异二聚体。 实验方法将依赖于有效的生产方法， 筛选特定的氨基酸置换突变。pro-pol-int编码 HIV-1的序列在E.在那里它被编码的 p66/p51异二聚体，其与 在受感染的人体细胞中产生。突变体设计将遵循 3-RT的三维X射线结构，并通过先前的突变分析 其鉴定了对折叠和/或稳定性至关重要的氨基酸残基。 为了确定对二聚化至关重要的相互作用，我们开发了一个简单的 可以对E. 表达RI的coil。p66亚基的C末端标记有六个 他的残留物，以便它可以保留在镍旋转柱。结合RT是 洗脱，通过Western印迹分析，并用抗RI单克隆抗体显色。 抗体的野生型RT显示p66和p51，p51保留在 在稳定的二聚体中与His标记的p66柱连接。突变体破坏了 二聚体显示很少或没有结合p51。 在关键残基处产生减少量的正确折叠的 将对活性RT进行研究，以确定残留物是否仅为关键 在折叠过程中，或者为了正确折叠的产品的稳定性。 预计参与折叠过程中重要步骤的残基 可以通过这种方式识别。相关结构的计算机建模 将进行RT（HIV-2、SIV和其他），以研究 相互作用的进化保守性对折叠、稳定性和 这些RT将在我们的表达系统中克隆，以允许 保守的关键相互作用的直接研究。

Description: The primary focus of this proposal is the identification of amino acid interactions that are critical for the formation of stable HIV-1 reverse transcriptase (RT) heterodimers. A detailed understanding of these interactions will potentially provide a basis for the rational design of new classes of therapeutically useful inhibitors for the processes involved in forming an active HIV-1 RT. The proposed experiments will distinguish between interactions critical for 1) protein folding, 2) stability of the folded protein structure, and 3) formation of the p66/p51 heterodimer. Approaches to the identification of peptide inhibitors for various stages in the synthesis and maturation of the RT heterodimer will also be investigated. The experimental approach will rely on efficient methods for production and screening of specific amino acid replacement mutations. The pro-pol-int coding sequence of HIV-1 is expressed in E. coli where it is processed by the encoded protease to produce p66/p51 heterodimer that is indistinguishable from that produced in the infected human cell. Mutant design will be guided by the 3-dimensional X-ray structure of the RT, and by previous mutational analysis which has identified amino acid residues critical for folding and/or stability. To identify interactions critical for dimerization, we have developed a simple assay for stable heterodimers which can be performed on crude extracts of E. coil expressing the RI. The C-terminus of the p66 subunit is tagged with six His residues so that it can be retained on nickel spin columns. The bound RT is eluted, analyzed by Western blotting, and visualized with an anti-RI monoclonal antibody. The wild-type RT shows both p66 and also p51 which is retained on the column in a stable dimer with the His tagged p66. Mutants that destabilize the dimer show little or no bound p51. Mutants at critical residues that produce reduced amounts of correctly folded active RT will be studied to determine whether the residue is critical only during the folding process, or for stability of the correctly folded product. It is expected that residues involved in important steps in the folding process can be identified in this way. Computer modeling of the structures of related RTs (HIV-2, SIV and others) will be performed to study the degree of evolutionary conservation of interactions critical for folding, stability, and dimerization of RT. These RTs will be cloned in our expression system to allow direct study of conserved critical interactions.